Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claims 1-36 are pending.
Applicant’s election of a method for selecting a multispecific antibody that read on (A) Fab/fab hetero Fc as the species of antibody construct, (B) CHO cell as the species of host cell, (C) A280 measurement SDS-PAD as the species of method of identify expression levels, (D) Octet as the measurement, (E) protein A as the method of purifying antibody, (F) LC-MS as the method of determining the percentage of correct and incorrect pairing, in the reply filed on May 1, 2026 is acknowledged. Because applicant did not distinctly and specifically point out the supposed errors in the restriction requirement, the election has been treated as an election without traverse (MPEP § 818.01(a)).
Claims 1-36, drawn to a method for selecting a multispecific antibody that read on (A) Fab/fab hetero Fc as the species of antibody construct, (B) CHO cell as the species of host cell, (C) A280 measurement SDS-PAD as the species of method of identify expression levels, (D) Octet as the measurement, (E) protein A as the method of purifying antibody, (F) LC-MS as the method of determining the percentage of correct and incorrect pairing, are being acted upon in this Office Action.
Priority
Applicant’ claim priority to provisional application 63/088,972, filed Oct 7, 2020, is acknowledged.
Information Disclosure Statement
The information disclosure statements (IDS) submitted on December 11, 2025, September 17, 2024 have been considered by the examiner and an initialed copy of the IDS is included with this Office Action.
The listing of references in the specification at pages 64-66 is not a proper information disclosure statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be incorporated into the specification but must be submitted in a separate paper." Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered.
Drawings
The drawings filed on April 7, 2023 are acceptable.
Specification
The preliminary amendment to the specification filed on April 7, 2023 has been entered.
The lengthy specification has not been checked to the extent necessary to determine the presence of all possible minor errors. Applicant's cooperation is requested in correcting any errors of which applicant may become aware in the specification.
Claim objection
Claims 1, 9 are objected to because of the following informality: the preamble of claim 1 or 9 recites “a method for selecting a multispecific antibody construct”, but ends with “in order to identify optimal pairing of the three heavy chain CDRs that specifically bind a first antigen and the light CDRs of (c)(iii) with the optimal pairing of the three heavy chain CDRs that specifically bind a second antigen and the same light chain CDRs of (c)(iii)”.
Claim 5 is objected to because of the following informality: “(HEK293”)baby” should have been “(HEK293”) baby”.
Claim 7 is objected to because of the following informality: the claim uses the abbreviation “SPR”, without first defining it. To clarify the claim, applicant should first spell out the full term before using an abbreviation. Given the subject matter of the specification, the examiner presumes that "SPR" stands for “Surface Plasmon Resonance (SPR)”. Appropriate correction isrequired.
Claim 8 is objected to because of the following informality: “and” at line 2 should have been “or”.
Claim 9 is objected to because of the following informality: “a multispecific antibody construct module” at line 18 is inconsistent with the “multispecific antibody construct” at line 1.
Claims 11, 19 are objected to because of the following informality: the preamble of claim 11 or 19 recites “a method for selecting a multispecific antibody construct”, but ends with “in order to identify optimal pairing of three heavy chain CDRs that specifically and three light chain CDRs that specifically bind a first antigen with three heavy chain CDRs and three light chain CDRs that specifically bind a second antigen”.
Claims 2, and 12 are objected to because of the following informality: “a first antigen” should have been “the first antigen” when referring back to preceding claim.
Claims 3, and 13 are objected to because of the following informality: “a first antigen” should have been “the second antigen” when referring back to preceding claim.
Claim 18 is objected to because of the following informality: “and” at line 2 should have been “or”.
Claims 21, 27 are objected to because of the following informality: the preamble of claim 21 or 27 recites “a method for selecting a multispecific antibody construct”, but ends with “in order to identify the optimal module for pairing of the three heavy chain CDRs that specifically bind a first antigen and the light chain CDRs of (c)(iii) with the three heavy chain CDRs that specifically bind a second antigen and the same light chain CDRs of (c)(iii)”.
Claim 25 is objected to because of the following informality: “and” at line 3 should have been “or”.
Claim 26 is objected to because of the following informality: “resins, and affinity tag” at line 2 should have been “resins, or affinity tag”.
Claim 27 is objected to because of the following informality: “and” is missing between step (fii) and (g).
Claim 29 is objected to because of the following informalities:
“antibody construct module” at line 9 is inconsistent with the “multispecific antibody construct” at line 1.
“different modules” at line 10 is inconsistent with the “multispecific antibody construct” at line 1.
“multispecific antibody construct module species” at line 17 is inconsistent with the “multispecific antibody construct” at line 1.
“CDRs of three light chain CDRs that do not specifically either the first antigen or second antigen” should have been “CDRs of three light chain CDRs that do not bind specifically to either the first antigen or second antigen”.
Claim 30 is objected to because of the following informality: “the multispecific antibody construct modules” should have been “the multispecific antibody construct”.
Claim 34 is objected to because of the following informality: “and” at line 2 should have been “or”.
Claims 29 and 35 are objected to because of the following informality: the preamble of claim 29 or 35 recites “a method for selecting a multispecific antibody construct”, but ends with “in order to identify the optimal multispecific antibody construct module for pairing of the three heavy chain CDRs and three light chain CDRs that specifically bind a first antigen with the three heavy chain CDRs and three light chain CDRs that specifically bind a second antigen”.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1, 4, 7, 10, 14, 17, 19, 21, 25, 27, 28, 29, 33 and 35 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which applicant regards as the invention.
The recitation of “one type of multispecific antibody construct module” in claim 1 is indefinite and ambiguous. One of ordinary skill in the art would not be reasonably apprised of the scope of the invention.
Claim 4 recites the limitation "the multispecific antibody construct modules" in claim 1. There is insufficient antecedent basis for this limitation in the claim. Deleting “modules” in claim 4 would obviate this rejection.
Claims 7, 25 recite trademark/trade names “Carterra LSA” and “Forte Bio”. Where a trademark or trade name is used in a claim as a limitation to identify or describe a particular material or product, the claim does not comply with the requirements of 35 U.S.C. 112, second paragraph. See Ex parte Simpson, 218 USPQ 1020 (Bd. App. 1982). The claim scope is uncertain since the trademark or trade name cannot be used properly to identify any particular material or product. A trademark or trade name is used to identify a source of goods, and not the goods themselves. Thus, a trademark or trade name does not identify or describe the goods associated with the trademark or trade name. In the present case, the trademark/trade name is used to identify/describe said assays and, accordingly, the identification/description is indefinite.
Claim 10 recites the limitation “the multispecific antibody construct modules” in claim 9. There is insufficient antecedent basis for this limitation in the claim. Amending claim 10 to recite “The method according to claim 9, wherein the multispecific antibody construct is selected from the group consisting of Fab/Fab hetero Fc…and Fab-scFv-Fc.” would obviate this rejection.
Claim 14 recites the limitation “the multispecific antibody construct module” in claim 11. There is insufficient antecedent basis for this limitation in the claim.
Claim 17 recites the limitation “the percentage of correct and incorrect multispecific antibody construct modality species” in claim 11. There is insufficient antecedent basis for this limitation in the claim.
The recitation of “a multispecific antibody construct module” in claim 19 is indefinite and ambiguous. One of ordinary skill in the art would not be reasonably apprised of the scope of the invention.
Regarding claim 21, the recitation of “more than one type of multispecific construct module” renders the claim indefinite because it is not clear how many type of multispecific construct modules and what they are. One of ordinary skill in the art would not be reasonably apprised of the scope of the invention.
Regarding claim 27, the recitation of “more than one type of multispecific construct module” renders the claim indefinite because it is not clear how many type of multispecific construct modules and what are the modules. One of ordinary skill in the art would not be reasonably apprised of the scope of the invention.
Claim 28 recites the limitation “the multispecific antibody construct modules” in claim 27. There is insufficient antecedent basis for this limitation in the claim.
Regarding claims 29 and 35, the recitation of “more than one type of multispecific construct module…different modules” renders the claim indefinite because it is not clear how many type of multispecific construct modules, what the modules are and how they are different from each other. One of ordinary skill in the art would not be reasonably apprised of the scope of the invention.
Claim 33 recites the limitation “the percentage of correct and incorrect multispecific antibody construct modality species” in claim 29. There is insufficient antecedent basis for this limitation in the claim.
Claim rejections under - 35 U.S.C. 112
The following is a quotation of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), first paragraph:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1-36 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for pre-AIA the inventor(s), at the time the application was filed, had possession of the claimed invention.
The Written Description Guidelines for examination of patent applications indicates, “the written description requirement for a claimed genus may be satisfied through sufficient description of a representative number of species by actual reduction to practice, or by disclosure of relevant, identifying characteristics, i.e., structure or other physical characteristics and/or other chemical properties, by functional characteristics coupled with a known or disclosed correlation between function and structure, or by a combination of such identifying characteristics, sufficient to show applicant was in possession of the claimed genus.” (see MPEP 2163).
Claim 1 encompasses a method for selecting a multispecific antibody construct, the method comprising:
(a) obtaining a plurality of antibody Fab fragments (elected species), scFvs, or a combination thereof, wherein each Fab fragment and scFv comprises three heavy chain CDRs and three light chain CDRs that specifically bind a first antigen;
(b) obtaining a plurality of antibody Fab fragments (species), scFvs, or a combination thereof, wherein each Fab fragment and scFv comprises three heavy chain CDRs and three light chain CDRs that specifically bind a second antigen;
(c) cloning into a vector:
(i) the CDRs of the three heavy chain CDRs that specifically bind the first antigen,
(ii) the CDRs of the three heavy chain CDRs that specifically bind the second antigen, and
(iii) three light chain CDRs selected from the group consisting of
(a) the CDRs of the three light chain CDRs that specifically bind the first antigen,
(b) the CDRs of the three light chain CDRs that specifically bind the second antigen, and
(c) CDRs of three light chain CDRs that do not specifically bind either the first antigen or the second antigen; wherein the vector(s) encode(s) for one type of multispecific antibody construct module and a plurality of vectors are generated that encode for a plurality of multispecific antibody constructs comprising the heavy chain CDRs that bind to each antigen and the light chain CDRs of (c) (iii);
(d) expressing each multispecific antibody construct in a mammalian host cell;
(e) purifying each multispecific antibody construct;
(f)(i) measuring the expression levels of each multispecific antibody construct and (ii) measuring the binding affinity of each multispecific antibody construct to the first antigen and the second antigen, wherein steps (f)(i) and (f)(ii) can be performed simultaneously or in any order; and
(g) comparing the expression levels of (f)(i) and the binding affinities of (f)(ii) for each multispecific antibody construct in order to identify the optimal pairing of the three heavy chain CDRs that specifically bind a first antigen and the light chain CDRs of (c)(iii) with the optimal pairing of the three heavy chain CDRs that specifically bind a second antigen and the same light chain CDRs of (c)(iii).
Claim 2 encompasses the method according to claim 1, wherein the plurality of antibody Fab fragments, scFvs, or a combination thereof, wherein each Fab fragment and scFv comprise three heavy chain CDRs and three light chain CDRs that specifically bind a first antigen is selected from the group consisting of at least two antibody Fab fragments, scFvs, or a combination thereof; at least three antibody Fab fragments, scFvs, or a combination thereof; at least four antibody Fab fragments, scFvs, or a combination thereof; at least five antibody Fab fragments, scFvs, or a combination thereof; at least six antibody Fab fragments, scFvs, or a combination thereof; at least seven antibody Fab fragments, scFvs, or a combination thereof; at least eight antibody Fab fragments, scFvs, or a combination thereof; at least nine antibody Fab fragments, scFvs, or a combination thereof; and at least ten antibody Fab fragments, scFvs, or a combination thereof.
Claim 3 encompasses the method according to claim 1, wherein the plurality of antibody Fab fragments, scFvs, or a combination thereof, wherein each Fab fragment and scFv comprise three heavy chain CDRs and three light chain CDRs that specifically bind a second antigen is selected from the group consisting of at least two antibody Fab fragments, scFvs, or a combination thereof; at least three antibody Fab fragments, scFvs, or a combination thereof; at least four antibody Fab fragments, scFvs, or a combination thereof; at least five antibody Fab fragments, scFvs, or a combination thereof; at least six antibody Fab fragments, scFvs, or a combination thereof; at least seven antibody Fab fragments, scFvs, or a combination thereof; at least eight antibody Fab fragments, scFvs, or a combination thereof; at least nine antibody Fab fragments, scFvs, or a combination thereof; and at least ten antibody Fab fragments, scFvs, or a combination thereof.
Claim 4 encompasses the method according to claim l, wherein the multispecific antibody construct modules include at least two of the modules selected from the group consisting of Fab/Fab hetero Fc (elected species), scFab/scFab hetero Fc, Fab/scFv hetero Fc, Fab/Fab-scFv hetero Fc, Fab/scFv-Fab hetero Fc, Fab/Fab hetero Fc-scFv, IgG-Fab, scFab-Fc-Fab, IgG-scFv, scFv-IgG, and Fab-scFv-Fc.
Claim 5 encompasses the method according to claim l, wherein the mammalian host cell is selected from the group consisting of Chinese hamster ovary ("CHO") cells (elected species), monkey kidney CV1 line transformed by SV40 ("COS-7"); human embryonic kidney line HEK293-6E ("HEK293-6E"); human embryonic kidney line 293 ("HEK293")baby hamster kidney cells ("BHK"); mouse sertoli cells Application No.: To Be Assigned (Docket No.: A-2696-USO2-PCT) Paper Filed: April 7, 2023 ("TM4"); monkey kidney cells ("CV1"); African green monkey kidney cells ("VERO-76"); human cervical carcinoma cells ("HELA"); canine kidney cells ("MDCK"); buffalo rat liver cells ("BRL"); human lung cells ("W138"); human hepatoma cells ("Hep G2"); mouse mammary tumor ("MMT"); TRI cells; MRC 5 cells: and FS4 cells.
Claim 6 encompasses the method according to claim 1, wherein the expression levels are determined by a method selected from the group consisting of A280 measurement SDS-PAGE (elected species), microchip capillary electrophoresis (MCE), Bradford assay, and bicinchoninic acid (BCA) assay.
Claim 7 encompasses the method according to claim 1, wherein binding affinity of each multispecific antibody construct to the first antigen and the second antigen is measured using Octet (elected species), Forte Bio, Carterra LSA, SPR and Flow cytometry.
Claim 8 encompasses the method according to claim l, wherein each multispecific antibody construct is purified by Protein A (elected species), Lambda and Kappa resins, and affinity tag purification.
Claim 9 encompasses a method for selecting a multispecific antibody construct, the method comprising:
(a) obtaining a plurality of at least two antibody Fab fragments (elected species), scFvs, or a combination thereof, wherein each Fab fragment and scFv comprises three heavy chain CDRs and three light chain CDRs that specifically bind a first antigen;
(b) obtaining a plurality of at least two antibody Fab fragments, scFvs, or a combination thereof, wherein each Fab fragment and scFv comprises three heavy chain CDRs and three light chain CDRs that specifically bind a second antigen;
(c) cloning into a vector: (i) the CDRs of the three heavy chain CDRs that specifically bind the first antigen, (ii) the CDRs of the three heavy chain CDRs that specifically bind the second antigen, and (iii) three light chain CDRs selected from the group consisting of (a) the CDRs of the three light chain CDRs that specifically bind the first antigen,(b) the CDRs of the three light chain CDRs that specifically bind the second antigen, and (c) CDRs of three light chain CDRs that do not specifically either the first antigen or the second antigen; wherein the vector(s) encode(s) for a multispecific antibody construct module and a plurality of vectors are generated that encode for a plurality of multispecific antibody constructs comprising the heavy chain CDRs that bind to each antigen and the light chain CDRs of (c)(iii);
(d) expressing each multispecific antibody construct in a mammalian host cell, wherein the mammalian host cell is selected from the group consisting of HEK293-6E cells and CHO cells (elected species);
(e) purifying each multispecific antibody construct using Protein A chromatography;
(f)(i) measuring the expression levels of each multispecific antibody construct using A280 measurement and (ii) measuring the binding affinity of each multispecific antibody construct to the first antigen and the second antigen using Octet, wherein steps (f)(i) and (f)(ii) can be performed simultaneously or in any order; and
(g) comparing the expression levels of (f)(i) and the binding affinities of (f)(ii) for each multispecific antibody construct in order to identify the optimal pairing of the three heavy chain CDRs that specifically bind a first antigen and the light chain CDRs of (c)(iii) with the optimal pairing of the three heavy chain CDRs that specifically bind a second antigen and the same light chain CDRs of (c)(iii).
Claim 10 encompasses the method according to claim 9, wherein the multispecific antibody construct modules include at least two of the modules selected from the group consisting of Fab/Fab hetero Fc (elected species), scFab/scFab hetero Fc, Fab/scFv hetero Fc, Fab/Fab-scFv hetero Fc, Fab/scFv-Fab hetero Fc, Fab/Fab hetero Fc-scFv, IgG-Fab, scFab-Fc-Fab, IgG-scFv, scFv-IgG, and Fab-scFv-Fc.
Claim 11 encompasses a method for selecting a multispecific antibody construct, the method comprising:
(a) obtaining a plurality of antibody Fab fragments, scFvs, or a combination thereof, wherein each Fab fragment and scFv comprises three heavy chain CDRs and three light chain CDRs that specifically bind a first antigen;
(b) obtaining a plurality of antibody Fab fragments, scFvs, or a combination thereof, wherein each Fab fragment and scFv comprises three heavy chain CDRs and three light chain CDRs that specifically bind a second antigen;
(c) cloning the CDRs of the two pluralities into a vector(s) that encode(s) for a multispecific antibody construct module and a plurality of vectors are generated that encode a plurality of multispecific antibody constructs comprising the CDRs that bind to each antigen;
(d) expressing each multispecific antibody construct in a mammalian host cell;
(e) purifying each multispecific antibody construct;
(f)(i) measuring the expression levels of each multispecific antibody construct and (ii) calculating the percent of correct and incorrect multispecific antibody construct module species produced, wherein steps (f)(i) and (f)(ii) can be performed simultaneously or in any order; and
(g) comparing the expression levels of (f)(i) and the percentage of correct and incorrect multispecific antibody construct module species of (f)(ii) for each multispecific antibody construct in order to identify the optimal pairing of three heavy chain CDRs and three light chain CDRs that specifically bind a first antigen with three heavy chain CDRs and three light chain CDRs that specifically bind a second antigen.
Claim 12 encompasses the method according to claim 11, wherein the plurality of antibody Fab fragments, scFvs, or a combination thereof, wherein each Fab fragment and scFv comprise three heavy chain CDRs and three light chain CDRs that specifically bind a first antigen is selected from the group consisting of at least two antibody Fab fragments, scFvs, or a combination thereof; at least three antibody Fab fragments, scFvs, or a combination thereof; at least four antibody Fab fragments, scFvs, or a combination thereof; at least five antibody Fab fragments, scFvs, or a combination thereof; at least six antibody Fab fragments, scFvs, or a combination thereof; at least seven antibody Fab fragments, scFvs, or a combination thereof; at least eight antibody Fab fragments, scFvs, or a combination thereof; at least nine antibody Fab fragments, scFvs, or a combination thereof; and at least ten antibody Fab fragments, scFvs, or a combination thereof.
Claim 13 encompasses the method according to claim 11 or claim 12, wherein the plurality of antibody Fab fragments, scFvs, or a combination thereof, wherein each Fab fragment and scFv comprise three heavy chain CDRs and three light chain CDRs that specifically bind a second antigen is selected from the group consisting of at least two antibody Fab fragments, scFvs, or a combination thereof; at least three antibody Fab fragments, scFvs, or a combination thereof; at least four antibody Fab fragments, scFvs, or a combination thereof; at least five antibody Fab fragments, scFvs, or a combination thereof; at least six antibody Fab fragments, scFvs, or a combination thereof; at least seven antibody Fab fragments, scFvs, or a combination thereof; at least eight antibody Fab fragments, scFvs, or a combination thereof; at least nine antibody Fab fragments, scFvs, or a combination thereof; and at least ten antibody Fab fragments, scFvs, or a combination thereof.
Claim 14 encompasses the method according to claim 11, wherein the multispecific antibody construct module is selected from the group consisting of Fab/Fab hetero Fc (elected species), scFab/scFab hetero Fc, Fab/scFv hetero Fc, Fab/Fab-scFv hetero Fc, Fab/scFv-Fab hetero Fc, Fab/Fab hetero Fc-scFv, IgG-Fab, scFab-Fc-Fab, IgG-scFv, scFv-IgG, and Fab-scFv-Fc.
Claim 15 encompasses the method according to claim 11, wherein the mammalian host cell is selected from the group consisting of Chinese hamster ovary ("CHO") cells (elected species), monkey kidney CV1 line transformed by SV40 ("COS-7"); human embryonic kidney line HEK293-6E ("HEK293-6E"); human embryonic kidney line 293 ("HEK293"); baby hamster kidney cells ("BHK"); mouse sertoli cells ("TM4"); monkey kidney cells ("CV1"); African green monkey kidney cells ("VERO- 76"); human cervical carcinoma cells ("HELA"); canine kidney cells ("MDCK"); buffalo rat liver cells ("BRL"); human lung cells ("W138"); human hepatoma cells ("Hep G2"); mouse mammary tumor ("MMT"); TRI cells; MRC 5 cells: and FS4 cells.
Claim 16 encompasses the method according to claim 11, wherein the expression levels are determined by a method selected from the group consisting of A280 measurement SDS-PAGE (elected species), microchip capillary electrophoresis (MCE), Bradford assay, and bicinchoninic acid (BCA) assay.
Claim 17 encompasses the method according to claim 11, wherein the percentage of correct and incorrect multispecific antibody construct modality species is determined by a method selected from the group consisting of liquid chromatography-mass spectrometry ("LC-MS") (elected species), Caliper, HPLC SEC, SDS-PAGE, and microchip capillary electrophoresis ("MCE").
Claim 18 encompasses the method according to claim 11, wherein each multispecific antibody construct is purified by Protein A (elected species), Lambda and Kappa resins, and affinity tag purification.
Claim 19 encompasses a method for selecting a multispecific antibody construct, the method comprising:
(a) obtaining a plurality of at least two antibody Fab fragments, scFvs, or a combination thereof, wherein each Fab fragment and scFv comprises three heavy chain CDRs and three light chain CDRs that specifically bind a first antigen;
(b) obtaining a plurality of at least two antibody Fab fragments, scFvs, or a combination thereof, wherein each Fab fragment and scFv comprises three heavy chain CDRs and three light chain CDRs that specifically bind a second antigen;
(c) cloning the CDRs of the two pluralities into a vector(s) that encode(s) for a multispecific antibody construct module and a plurality of vectors are generated that encode a plurality of multispecific antibody constructs that bind to each antigen; It is within the purview of one of ordinary skilled in the art to clone more than one CDRs of the heavy chain comprising three CDRs that bind to a first antigen, clone more than one CDRs of the heavy chain comprising three CDRs that bind to a second antigen, more than one CDRs of the light chain comprising three CDRs that bind to a first antigen, more than one CDRs of the light chain comprising three CDRs that bind to a second antigen, or CDRs of a common light chain comprising three CDRs in a vector or vectors for a Fab library, e.g., screening, and subcloning into different vectors for each bispecific antibodies expression.
(d) expressing each multispecific antibody construct in a mammalian host cell, wherein the mammalian host cell is selected from the group consisting of HEK293-6E cells and CHO cells (elected species);
(e) purifying each multispecific antibody construct using Protein A chromatography (elected species);
(f)(i) measuring the expression levels of each multispecific antibody construct using A280 measurement and (ii) calculating the percent of correct and incorrect multispecific antibody construct module species produced using liquid chromatography-mass spectrometry ("LC-MS") (elected species), wherein steps (f)(i) and (f)(ii) can be performed simultaneously or in any order; and
(g) comparing the expression levels of (f)(i) and the percentage of correct and incorrect multispecific antibody construct module species of (f)(ii) for each multispecific antibody construct in order to identify the optimal pairing of three heavy chain CDRs and three light chain CDRs that specifically bind a first antigen with three heavy chain CDRs and three light chain CDRs that specifically bind a second antigen.
Claim 20 encompasses the method according to claim 19, wherein the multispecific antibody construct module is selected from the group consisting of Fab/Fab hetero Fc (elected species), scFab/scFab hetero Fc, Fab/scFv hetero Fc, Fab/Fab-scFv hetero Fc, Fab/scFv-Fab hetero Fc, Fab/Fab hetero Fc-scFv, IgG-Fab, scFab-Fc-Fab, IgG- scFv, scFv-IgG, and Fab-scFv-Fc.
Claim 21 encompasses a method for selecting a multispecific antibody construct, the method comprising:
(a) obtaining a first antibody Fab fragment or scFv, wherein each Fab fragment or scFv comprises three heavy chain CDRs and three light chain CDRs that specifically bind a first antigen;
(b) obtaining a second antibody Fab fragment or scFv, wherein each Fab fragment or scFv comprises three heavy chain CDRs and three light chain CDRs that specifically bind a second antigen;
(c) cloning into a vector: (i) the CDRs of the three heavy chain CDRs that specifically bind the first antigen, (ii) the CDRs of the three heavy chain CDRs that specifically bind the second antigen, and (iii) three light chain CDRs selected from the group consisting of (a) the CDRs of the three light chain CDRs that specifically bind the first antigen, (b) the CDRs of the three light chain CDRs that specifically bind the second antigen, and (c) CDRs of three light chain CDRs that do not specifically either the first antigen or the second antigen; wherein the vector(s) encode(s) for more than one type of multispecific antibody construct module and a plurality of vectors are generated that encode for a plurality of multispecific antibody constructs of different modules comprising the heavy chain CDRs that bind to each antigen and the light chain CDRs of (c)(iii);
(d) expressing each multispecific antibody construct of the different modules in a mammalian host cell;
(e) purifying each multispecific antibody construct;
(f)(i) measuring the expression levels of each multispecific antibody construct and (ii) measuring the binding affinity of each multispecific antibody construct to the first antigen and the second antigen, wherein steps (f)(i) and (f)(ii) can be performed simultaneously or in any order; and
(g) comparing the expression levels of (f)(i) and the binding affinities of (f)(ii) for each multispecific antibody construct in order to identify the optimal module for pairing of the three heavy chain CDRs that specifically bind a first antigen and the light chain CDRs of (c)(iii) with the three heavy chain CDRs that specifically bind a second antigen and the same light chain CDRs of (c)(iii).
Claim 22 encompasses the method according to claim 21, wherein the multispecific antibody construct modules include at least two of the modules selected from the group consisting of Fab/Fab hetero Fc (elected species), scFab/scFab hetero Fc, Fab/scFv hetero Fc, Fab/Fab-scFv hetero Fc, Fab/scFv-Fab hetero Fc, Fab/Fab hetero Fc-scFv, IgG-Fab, scFab-Fc-Fab, IgG-scFv, scFv-IgG, and Fab-scFv-Fc.
Claim 23 encompasses the method according to claim 21, wherein the mammalian host cell is selected from the group consisting of Chinese hamster ovary ("CHO") cells (elected species), monkey kidney CV1 line transformed by SV40 ("COS-7"); human embryonic kidney line HEK293-6E ("HEK293-6E"); human embryonic kidney line 293 ("HEK293"); baby hamster kidney cells ("BHK"); mouse sertoli cells ("TM4"); monkey kidney cells ("CV1"); African green monkey kidney cells ("VERO- 76"); human cervical carcinoma cells ("HELA"); canine kidney cells ("MDCK"); buffalo rat liver cells ("BRL"); human lung cells ("W138"); human hepatoma cells ("Hep G2"); mouse mammary tumor ("MMT"); TRI cells; MRC 5 cells: and FS4 cells.
Claim 24 encompasses the method according to claim 21, wherein the expression levels are determined by a method selected from the group consisting of A280 measurement SDS-PAGE (elected species), microchip capillary electrophoresis (MCE), Bradford assay, and bicinchoninic acid (BCA) assay.
Claim 25 encompasses the method according to claim 21, wherein binding affinity of each multispecific antibody construct to the first antigen and the second antigen is measured using Octet (elected species), Forte Bio, Carterra LSA, SPR and Flow cytometry.
Claim 26 encompasses the method according to claim 21, wherein each multispecific antibody construct is purified by Protein A (elected species), Lambda and Kappa resins, and affinity tag purification.
Claim 27 encompasses a method for selecting a multispecific antibody construct, the method comprising:
(a) obtaining a first antibody Fab fragment or scFv, wherein each Fab fragment or scFv comprises three heavy chain CDRs and three light chain CDRs that specifically bind a first antigen;
(b) obtaining a second antibody Fab fragment or scFv, wherein each Fab fragment or scFv comprises three heavy chain CDRs and three light chain CDRs that specifically bind a second antigen;
(c) cloning into a vector: (i) the CDRs of the three heavy chain CDRs that specifically bind the first antigen, (ii) the CDRs of the three heavy chain CDRs that specifically bind the second antigen, and (iii) three light chain CDRs selected from the group consisting of (a) the CDRs of the three light chain CDRs that specifically bind the first antigen,(b) the CDRs of the three light chain CDRs that specifically bind the second antigen, and (c) CDRs of three light chain CDRs that do not specifically either the first antigen or the second antigen; wherein the vector(s) encode(s) for more than one type of multispecific antibody construct module and a plurality of vectors are generated that encode for a plurality of multispecific antibody constructs of different modules comprising the heavy chain CDRs that bind to each antigen and the light chain CDRs of (c)(iii);
(d) expressing each multispecific antibody construct in a mammalian host cell, wherein the mammalian host cell is selected from the group consisting of HEK293-6E cells and CHO cells (elected species);
(e) purifying each multispecific antibody construct using Protein A chromatography;
(f)(i) measuring the expression levels of each multispecific antibody construct using A280 measurement and (ii) measuring the binding affinity of each multispecific antibody construct to the first antigen and the second antigen using Octet,
(g) comparing the expression levels of (f)(i) and the binding affinities of (f)(ii) for each multispecific antibody construct in order to identify the optimal module for pairing of the three heavy chain CDRs that specifically bind a first antigen and the light chain CDRs of (c)(iii) with the three heavy chain CDRs that specifically bind a second antigen and the same light chain CDRs of (c)(iii).
Claim 28 encompasses the method according to claim 27, wherein the multispecific antibody construct modules include at least two of the modules selected from the group consisting of Fab/Fab hetero Fc (elected species), scFab/scFab hetero Fc, Fab/scFv hetero Fc, Fab/Fab-scFv hetero Fc, Fab/scFv-Fab hetero Fc, Fab/Fab hetero Fc-scFv, IgG-Fab, scFab-Fc-Fab, IgG-scFv, scFv-IgG, and Fab-scFv-Fc.
Claim 29 encompasses a method for selecting a multispecific antibody construct, the method comprising:
(a) obtaining a first antibody Fab fragment or scFv, wherein each Fab fragment or scFv comprises three heavy chain CDRs and three light chain CDRs that specifically bind a first antigen;
(b) obtaining a second antibody Fab fragment or scFv, wherein each Fab fragment or scFv comprises three heavy chain CDRs and three light chain CDRs that specifically bind a second antigen;
(c) cloning the CDRs of the first antibody Fab fragment or scFv and the second antibody Fab fragment or scFv into a vector(s), wherein the vector(s) encode(s) for more than one type of multispecific antibody construct module and a plurality of vectors are generated that encode for a plurality of multispecific antibody constructs of different modules comprising the CDRs that bind to each antigen;
(d) expressing each multispecific antibody construct in a mammalian host cell;
(e) purifying each multispecific antibody construct;
(f)(i) measuring the expression levels of each multispecific antibody construct and (ii) calculating the percent of correct and incorrect multispecific antibody construct module species produced, wherein steps (f)(i) and (f)(ii) can be performed simultaneously or in any order; and
(g) comparing the expression levels of (f)(i) and the percentage of correct and incorrect multispecific antibody construct module species of (f)(ii) for each multispecific antibody construct in order to identify the optimal multispecific antibody construct module for pairing of the three heavy chain CDRs and three light chain CDRs that specifically bind a first antigen with the three heavy chain CDRs and three light chain CDRs that specifically bind a second antigen.
Claim 30 encompasses the method according to claim 29, wherein the multispecific antibody construct modules include at least two of the modules selected from the group consisting of Fab/Fab hetero Fc (elected species), scFab/scFab Paper Filed: April 7, 2023 hetero Fc, Fab/scFv hetero Fc, Fab/Fab-scFv hetero Fc, Fab/scFv-Fab hetero Fc, Fab/Fab hetero Fc-scFv, IgG-Fab, scFab-Fc-Fab, IgG-scFv, scFv-IgG, and Fab-scFv-Fc.
Claim 31 encompasses the method according to claim 29, wherein the mammalian host cell is selected from the group consisting of Chinese hamster ovary ("CHO") cells (elected species), monkey kidney CV1 line transformed by SV40 ("COS-7"); human embryonic kidney line HEK293-6E ("HEK293-6E"); human embryonic kidney line 293 ("HEK293"); baby hamster kidney cells ("BHK"); mouse sertoli cells ("TM4"); monkey kidney cells ("CV1"); African green monkey kidney cells ("VERO- 76"); human cervical carcinoma cells ("HELA"); canine kidney cells ("MDCK"); buffalo rat liver cells ("BRL"); human lung cells ("W138"); human hepatoma cells ("Hep G2"); mouse mammary tumor ("MMT"); TRI cells; MRC 5 cells: and FS4 cells.
Claim 32 encompasses the method according to claim 29, wherein the expression levels are determined by a method selected from the group consisting of A280 measurement SDS-PAGE (elected species), microchip capillary electrophoresis (MCE), Bradford assay, and bicinchoninic acid (BCA) assay.
Claim 33 encompasses the method according to claim 29, wherein the percentage of correct and incorrect multispecific antibody construct modality species is determined by a method selected from the group consisting of liquid chromatography-mass spectrometry ("LC-MS") (elected species), Caliper, HPLC SEC, SDS-PAGE, and microchip capillary electrophoresis ("MCE").
Claim 34 encompasses the method according to claim 29, wherein each multispecific antibody construct is purified by Protein A, Lambda and Kappa resins, and affinity tag purification.
Claim 35 encompasses a method for selecting a multispecific antibody construct, the method comprising:
(a) obtaining a first antibody Fab fragment or scFv, wherein each Fab fragment or scFv comprises three heavy chain CDRs and three light chain CDRs that specifically bind a first antigen;
(b) obtaining a second antibody Fab fragment or scFv, wherein each Fab fragment or scFv comprises three heavy chain CDRs and three light chain CDRs that specifically bind a second antigen;
(c) cloning the CDRs of the first antibody Fab fragment or scFv and the second antibody Fab fragment or scFv into a vector(s), wherein the vector(s) encode(s) for more than one type of multispecific antibody construct module and a plurality of vectors are generated that encode for a plurality of multispecific antibody constructs of different modules comprising the CDRs that bind to each antigen;
(d) expressing each multispecific antibody construct in a mammalian host cell, wherein the mammalian host cell is selected from the group consisting of HEK293-6E cells and CHO cells (elected species);
(e) purifying each multispecific antibody construct using Protein A chromatography;
(f)(i) measuring the expression levels of each multispecific antibody construct using A280 and (ii) calculating the percent of correct and incorrect multispecific antibody construct module species produced using liquid chromatography-mass spectrometry ("LC-MS"), wherein steps (f)(i) and (f)(ii) can be performed simultaneously or in any order; and
(g) comparing the expression levels of (f)(i) and the percentage of correct and incorrect multispecific antibody construct module species of (f)(ii) for each multispecific antibody construct in order to identify the optimal multispecific antibody construct module for pairing of the three heavy chain CDRs and three light chain CDRs that specifically bind a first antigen with the three heavy chain CDRs and three light chain CDRs that specifically bind a second antigen.
Claim 36 encompasses the method according to claim 35, wherein the multispecific antibody construct modules include at least two of the modules selected from the group consisting of Fab/Fab hetero Fc (elected species), scFab/scFab hetero Fc, Fab/scFv hetero Fc, Fab/Fab-scFv hetero Fc, Fab/scFv-Fab hetero Fc, Fab/Fab hetero Fc-scFv, IgG-Fab, scFab-Fc-Fab, IgG-scFv, scFv-IgG, and Fab-scFv-Fc.
The specification discloses:
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Description of Chain Selectivity Assessment Screening Methods
[0389] To facilitate the development of IgG-like Bispecifics, a high-throughput screening process, CSA, to evaluate HC/LC selectivity (FIG. 2A), is envisioned. Since the antibody HC is only secreted when bound to the LC.sup.21,22, the measure of expression level of a given HC/LC pair may correlate to the HC/LC pairing efficiency. Starting from 2 panels of parental antibodies (anti-Target-A and anti-Target-B), high-throughput CSA was deployed in 2 different scenarios (competition and non-competition) to evaluate the specificity in the assembly of Hetero-Fcs (4 chains) and to identify promiscuous LCs for cLC Hetero-Fcs (3 chains), respectively (FIGS. 2A and B). The cCSA experiment mimics a co-expression scenario of 4 different chains (2×HCs and 2×LCs), resulting in all possible combinations between anti-Target-A and anti-Target-B parental antibodies. The expressed Hetero-Fcs were purified from conditioned medium with ProA beads and the percentage of correct HC/LC pairing was quantitated through high-resolution liquid chromatography-mass spectrometry (LC-MS) (FIGS. 2B and 15). It's worth noting that 2 out the 4 Hetero-Fc species may show the same MW, with one of them representing a scenario where both LCs (LCA and LCB) have a perfect cross pair with the opposite non-cognate HCs (FIG. 2B). Although this LC-MS cannot distinguish such species, this scramble of LCs is rarely seen even after limited proteolysis, strongly suggesting that the correct MW detected by LC-MS indicates correct HC/LC pairing. The high percentage of correct species is an indication of the preference for cognate HC/LC pairing over the non-cognate. The antibody combinations with high levels of correct species will be selected as the optimal building blocks for Bispecifics that require correct/specific HC/LC pairing (FIG. 2B). As for the ncCSA experiment, every single HC from anti-Target-A pairs with every LC from anti-Target-B at a 1:1 ratio, and vice versa. High expression levels (comparable to HC/LC cognate pair or higher) measured by ProA capture will indicate that LCs pair well and allow the non-cognate HCs to fold and to be secreted (FIG. 2B). In contrast, low expression levels suggest that the VH/VL interface of these chain(s) are specific to their cognate interfaces and as result cannot accommodate others. Next, using the same ProA purified material, a second high-throughput step was integrated to characterize the binding affinity using ForteBio Octet. This protocol enables a quick (less than 5 weeks) and efficient tool to screen and identify rare and valuable LCs that can be used as building blocks for cLC bispecific Abs.
Identification of Low-Crosspairing Antibody Combinations with Competition CSA Method
[0390] To validate the cCSA method, 2 panels of parental antibodies, 8× anti-Target-A Abs (A1-A8) and 4× anti-Target-B (B1-B4), were selected. Mutations were introduced to drive Heavy-Heavy chain pairing and combined antibodies from each target, resulting in 32 combinations for evaluation. Following the purification of the secreted IgGs from conditioned media, the expression levels of the combinations and parental mAbs were measured by A280 (FIGS. 3A and 16). Notably, parental mAbs demonstrated a high variation in expression levels ranging from 50 to 250 mg/L. Surprisingly, most of the antibody combinations expressed at levels higher than 100 mg/L with few exceptions (A3×B1, A3×B3, A3×B4 and A6×B1), which may be related to the low expressing parents A3 and B1. To identify whether the species assembled and secreted have correct HC/LC pairing, non-reducing LC-MS was used to analyze the ProA purified proteins (exemplified in FIG. 3B). By using the Fc region of the molecules for purification with ProA beads, HC containing molecules only was selected for and all other species (e.g. LC dimers) are discarded. Although the LC-MS analysis cannot confirm whether the 2 pairs of cognate HC/LC are correctly paired, it can determine whether each species has a copy of each of the 4 chains. While not definitive, this is a required condition towards the assembly and production of Hetero-Fcs. Therefore, the percentage for each of these 3 possible HC/LC scenarios was calculated: 1×HC.sub.A+1×HC.sub.B+1×LC.sub.A+1×LC.sub.B, 1×HC.sub.A+1×HC.sub.B+2×LC.sub.A+0×LC.sub.B and 1×HC.sub.A+1×HC.sub.B+0×LC.sub.A+2×LC.sub.B, all containing HC heterodimers (FIG. 3C). Although two combinations (A2×B3 and A4×B3) failed in LC-MS analysis due to small MW difference between two LCs (<60 Da), successful quantitation of the IgG species in all other 30 combinations was achieved. Furthermore, although Fc CPMs in the Ab combinations was deployed to enhance HC heterodimerization, there were still small amounts of homodimers and/or ½ Abs (FIG. 8). However, since the focus of this study is on HC/LC pairing, those species were excluded from further analysis. Interestingly, the data showed that in about half of the molecules tested (17/32), the percentage of the desired species (1×HC.sub.A+1×LC.sub.A+1×HC.sub.B+1×LC.sub.B) detected was ≥50% of the total species present in the ProA purified samples (FIG. 3C). Surprisingly, in 2 combinations (A6×B3 and A6×B4) the percentage of the correct product was >75%. In contrast, the ProA purified samples of the remaining 13 combinations contained <50% of the species of interest, with 5 of those combinations (A1×B1, A2×B1, A3×B2, A4×B1 and A7×B1) containing less than 25% of the desired species (FIG. 3C). In most cases (23/32) only 1/2 LCs appeared in the undesired product (1×HC.sub.A+1×HC.sub.B+2×LC.sub.A+0×LC.sub.B and 1×HC.sub.A+1×HC.sub.B+0×LC.sub.A+2×LC.sub.B), suggesting that either one of the LCs is overexpressing or that LC.sub.A and LC.sub.B may compete during expression and molecule assembly leading to a complete or partial suppression of the other (FIG. 3C). Thus, with this information, the cCSA method can efficiently screen and identify suitable combinations of parental mAbs with native properties that enable the correct assembly of the Hetero-Fcs upon expression in a single cell. This allows for deselection of candidates while directing efforts towards those with more promising characteristics, resulting in time and resources savings.
Low-Cross pairing Antibody Combinations are Good Candidates for Making 4-Chain Hetero-Fcs
[0391] In the cCSA method described above, more than half (17/32) of the combinations showed high level (≥50%) of desired species (1×HC.sub.A+1×LC.sub.A+1×HC.sub.B+1×LC.sub.B) (FIG. 3C). However, to assess whether this high-throughput method was truly predictive of correct cognate HC/LC pairing, it was decided to scale-up the 32 Hetero-Fcs and perform a 2-step purification with ProA followed by a cation exchange chromatography (CIEX). The goal was not only to quantify the final yields of the desired species but also to study the separation profile in the CIEX step. From the 32 Hetero-Fcs, 11 were successfully purified with a final purity >90% as determined by SEC and LC-MS (FIG. 4A and FIG. 8). Interestingly, the final yields for the parental mAbs, the building blocks for these Hetero-Fcs, appear to correlate with the yields of the resulting bispecific molecules. Indeed, Hetero-Fcs A1×B3, A1×B4, A4×B3 and A8×B3 displaying the highest protein yields all contain at least one of the high expressing parental mAbs A1, A4 and A8 (FIG. 4A). For final verification, the final pools with LC-MS purity >90% were evaluated for binding against Target-A and —B, critical to evaluate the cognate HC/LC pairing. The affinity of each arm in the bispecific molecule to its respective target was comparable to that of the parent mAbs (FIG. 17), confirming that all 11 Hetero-Fcs have correct HC/LC in both arms.
[0392] Overall, this data aligns well with the cCSA experiment (FIG. 4E). From the 11 successfully purified Hetero-Fcs, the percentage of correct HC/LC pairing predicted by LC-MS was high for 8 molecules (≥50% correct HC/LC species, A1×B3, A1×B4, A3×B3, A4×B2, A4×B4, A6×B2, A6×B4 and A8×1B3), good for A7×B4 (47.4% correct species) and unknown for A2×B3 and A4×B3 (indeterminate due to the small MW difference between chains). Most importantly, none of the molecules that showed low HC/LC pairing by CSA could be purified by CIEX (FIGS. 3C and 4A). Indeed, the percentage of correct species determined by cCSA was predictive of which Ab combinations resulted in successful Hetero-Fcs (ROC AUC 0.80, FIG. 4B). Furthermore, we also attempted to build correlation between the percentage of HC/LC pairing predicted by cCSA and the final yields. As shown in FIG. 4C, most of the combinations (12/13) with <50% correct HC/LC pairing by cCSA failed later during the CIEX purification. One exception was the A7×B4 molecule that while displaying 47.4% pairing, it showed an CIEX profile with suitable separation (FIG. 4D). Interestingly, CIEX could not purify 9 out of 17 molecules with >50% correct HC/LC pairing predicted by cCSA (FIG. 4C). Indeed, to illustrate this phenomenon we have selected the example of the A8×B4 Hetero-Fc that displayed 69.4% correct pairing in the cCSA experiments. This molecule exhibits a well-shaped peak that conceals the mispaired species in such a way that no resolution between the two different species (1×HC.sub.A+1×HC.sub.B+1×LC.sub.A+1×LC.sub.B and 1×HC.sub.A+1×HC.sub.B+2×LC.sub.A+0×LC.sub.B) is observed (FIG. 4D). Thus, while the cCSA method can predict high performing Hetero-Fc molecules based on the native VH/VL interface, properties not screened by cCSA (such as those influencing separation profiles on ion-exchange chromatography columns) also play a significant role in selecting Hetero-Fcs.
Screening for Common LCs with the Non-Competition CSA Method
[0393] As shown above, while some LCs pair preferably with their cognate HCs, other LCs can also bind to non-cognate HCs efficiently. If the resulting non-cognate HC/LC pair retains binding, such a LC could serve as a common LC (cLC). The ncCSA method is envisioned as an opportunistic approach to evaluate whether the parental mAbs offer such LCs (FIG. 2B). To demonstrate the efficacy of this method, two bispecific programs (A×B and C×B) with 8 anti-Target-A, 4 anti-Target-B and 10 anti-Target-C parental mAbs were selected. As shown in FIG. 9, every LC of anti-Target-B was paired with individual HCs from anti-Target-A or -C mAbs. Meanwhile, each HC of anti-Target-B was combined with individual LCs from different anti-Target-A or -C mAbs. The resulting 144 non-cognate HC/LC pairs were expressed together with 22 parental mAbs (control) in 293 6E and purified with ProA beads, followed by analysis with non-reducing SDS PAGE gel and A280 quantitation (FIG. 10). Further analysis of the ProA yields for these 144 non-cognate HC/LC pairs (FIG. 5A and FIG. 9) showed that only 38 of the 144 combinations (26.4%) displayed a significant reduction in expression levels (50% or lower relative to the controls), suggesting an overall widespread promiscuous behavior within HC/LC pairing. Of the remaining 106 combinations, 68 (47.2%) HC/LC non-cognate pairing molecules displayed higher expression levels than the corresponding parental mAb controls (FIG. 5A). However, many LCs were not broadly promiscuous. A closer look into 2 examples is useful to illustrate this phenomenon. When LCs-B1-4 were paired with HC-A7, protein expression was significantly lower when compared to the cognate LC-A7 (control) (FIG. 5B). In contrast, the expression levels of the same anti-Target-B LCs while paired with HC-A8 are comparable or higher than cognate LC-A8 (control). Thus, suggesting that the anti-Target-B LCs are promiscuous with respect to HC-A8 but not towards HC-A7, highlighting the role that HCs also play a role in determining LC cross pairing.
Binding Analysis Identified Common LC Hetero-Fc Candidates
[0394] Although well-expressing non-cognate Ab combinations are promising candidates to assemble cLC Bispecifics, expression levels alone provide no insight into function. To select for functional Bispecifics, selected promiscuous LCs (demonstrating 50% or higher expression levels relative to cognate controls during the ncCSA assay (FIG. 5A)) were assembled into a Hetero-Fc format with both the cognate and non-cognate HCs, containing Fc CPMs to promote HC dimerization (FIG. 5C). These cLC Hetero-Fcs were evaluated with a high-throughput binding assay to identify candidates that allow for binding to both targets. After high-throughput expression in 4 mL deep well blocks (DWB), with HEK293-6E cells, the cLC Hetero-Fcs were purified by ProA. The yields for 92 out of 106 of the molecules (86.8%), was ˜100 mg/L, which is comparable to the parental mAbs (FIG. 3A and FIG. 11), with only 14 cLC Hetero-Fcs (13.2%) showing ProA yields lower than 60 mg/L (FIGS. 5D and E). All molecules with cLC B1 showed a remarkably lower expression, suggesting that this LC may act as limiting factor in the overall expression of these Bispecifics (FIG. 5D). Indeed, the ProA yield for B1 Ab with 41.6 mg/L was the lowest among all parental mAbs used as building blocks for the generation of these Bispecifics (FIG. 3A). Although A3 and A6 parental mAbs also showed a relatively low ProA recovery (60.3 and 69.3 mg/L, respectively), the cLC Hetero-Fcs containing these 2 building blocks showed acceptable protein yields when combined with any B parental but B1, suggesting that in this case the non-cognate HCs may have rescued the expression levels (FIG. 5D). Another important observation is that the cLC Hetero-Fcs (A×B and C×B) also showed a ˜2-fold increase overall in correct pairing over the 4-chain Hetero-Fcs just after ProA purification, highlighting the impact of HC/LC pairing in the production levels of these molecules (FIG. 12).
[0395] For rapid binding screening, these single-step purified samples were then assessed by ForteBio Octet. To minimize interference by residual impurities, the cLC-Hetero-Fc molecules were first captured onto Streptavidin fiber optic biosensors with a biotinylated anti-human IgG Fc polyclonal antibody via the Fc region and soluble antigen-A, -B or -C were loaded for incubation. As expected, all cLC Hetero-Fcs displayed binding to their respective targets via the cognate HC/LC arm, with comparable affinity to the parental mAbs (FIG. 5F). 2 cLC Hetero-Fcs (A2×B4 and C4×B3), also showed detectable binding via the non-cognate HC/LC arm recognizing Target-A or -C(FIG. 5F). In the case of A2×B4, the B4 LC was paired with both HCs (A2 and B4), whereas for C4×B3 the HCs (C4 and B3) were both paired with the B3 LC. Of note, these cLCs were both generated against Target-B. Although this non-canonical binding is lower than the single-digit nM binding typically observed for parental mAbs (FIG. 13), it demonstrates how the ncCSA method provides a new opportunity to identify LCs with unique structural features allowing for highly efficient pairing with non-cognate HCs (FIG. 5G). Furthermore, rapid binding analysis can reveal those rare cLCs that also retain binding to a new epitope. Since the manufacturability of IgG-like Bispecifics is often challenging with production levels below that of mAbs.sup.23, the expression and purification properties of these cLC Hetero-Fcs was explored. To better mimic the scale and purification process required for therapeutic candidates, these 2 molecules were expressed in 250 mL HEK293-6E cells and subjected to a 2-step purification with ProA followed by CIEX to meet the purity target of >95%. Notably, the levels of protein secretion, by ProA, were over 2-fold higher for these 2 cLC Hetero-Fcs when compared to the parental mAbs (FIG. 14). More importantly, these cLC Hetero-Fcs showed a final yield comparable to or higher than the parental mAbs (FIG. 6A), all with over 97% purity of the desired species (FIG. 14). Moreover, these Bispecifics showed favorable CIEX profiles with correct species easily separated from the impurities (FIG. 6B). The binding assay was then repeated using the fully purified cLC Hetero-Fcs, to confirm their affinity for the respective antigens. As observed initially (FIG. 5F), these 2 molecules showed binding affinity via their non-cognate HC/LC arms to antigen-A or C while retaining the binding properties in the cognate arms to antigen-B (FIGS. 6C and S7). To validate the affinity measured for these cLC Hetero-Fcs, two hybrid IgGs composed of a non-cognate HC and LC each (HC-A2/LC-B4 and HC-C4/LC-B3) were expressed and purified. The comparable affinities of the hybrid molecules to antigen-A and —C via their non-cognate arms (FIG. 6D), further confirmed the cLC Hetero-Fcs binding. The binding signal for the hybrid IgGs was ˜2-fold higher than the signal observed for the non-cognate arm in the cLC Hetero-Fcs, which agrees with the number of binding sites present in these molecules (2 vs 1, respectively). Moreover, since neither of them seems to retain binding to antigen-B, it suggests that the binding capability of hybrid IgGs is mostly driven by HC CDRs but not LC. Inversely, to also exclude the possibility of non-specific binding to antigen-A or —C by the cognate arms in the cLC Hetero-Fcs, the binding for B4 and B3 parental mAbs was tested. As shown in FIG. 6E, B4 and B3 mAbs did not bind to these antigens, further demonstrating the binding detected for the non-cognate arm is not derived from a non-specific interaction between cognate arm and antigen-A or -C nor is the result of cLC alone.
[0396] Altogether, the ncCSA method successfully identified two rare LCs from a pool of mAbs that paired with non-cognate HCs showing mAb like productivity while allowing the HC in this non-cognate HC/LC arm to retain binding activity.
However, the specification does not describe methods of selecting any multispecific antibody construct as set forth in claims 1-36 by obtaining any scFvs or combination of Fab fragments and scFvs that binds to s first antigen or a second antigen in step (a) and (b). There are no examples of heavy and light chain pairing using single chain antibody comprising VH and VL to form IgG heterodimer bispecific antibody.
Regarding vectors encode for one type of multispecific antibody construct module (claims 1, 9, 11, 19, 21, 27, 29, 35), the specification discloses vector encoding each hetero-Fc combinations were transiently expressed in just HEK293-6E cells, see caption of FIG.3. All combinations of 2 HCs and 2 LCs are co-transfected into HEK293-6E cells, see caption of FIG. 2.
However, the specification does not disclose expressing each multispecific antibody construct module such as scFab/scFab hetero Fc, Fab/scFv hetero Fc, Fab/Fab-scFv hetero Fc, Fab/scFv-Fab hetero Fc, Fab/Fab hetero Fc-scFv, IgG-Fab, scFab-Fc-Fab, IgG-scFv, scFv-IgG, and Fab-scFv-Fc that bind to any “first antigen” and any “second antigen” to demonstrate possession of the genus at the time of filing.
At the time the invention was made, Shiraiwa (Methods 154: 10-20, 2019; PTO 1449) teaches that theoretically, a common L chain can be found by using antibody libraries or mice bearing a common L chain, as well as by the FR/CDR shuffling method. However, all methods of obtaining a common L chain sometimes produce a bispecific antibody that lacks sufficient antigen-binding affinity, see p. 18, right col. Further optimization would be necessary to improve various properties, such as pI modification, pH dependency for high-quality production. Further, complete control is still difficult by these methods, and how to analyze impurities ad how to establish quantitative methods are emerging issues in clinical development, see p. 11, right col.
Given the size of the genus of antibody heavy and light chains that bind to any undisclosed antigens in any multispecific antibody constructs (aka bispecific IgG antibody format), the specification at best describes a plan for making such multispecific antibody construct having the claimed attributes or limitations, and then identifying those that satisfy claim limitations, but mere “wish or plan” for obtaining claimed invention is not sufficient. Centocor Ortho Biotech Inc. v. Abbott Laboratories, 97 USPQ2d 1870 (Fed. Cir. 2011).
Vas-Cath Inc. v. Mahurkar, 19 USPQ2d 1111, makes clear that “applicant must convey with reasonable clarity to those skilled in the art that, as of the filing date sought, he or she was in possession of the invention. The invention is, for purposes of the written description inquiry, whatever is now claimed.” (See page 1117.) The specification does not “clearly allow persons of ordinary skill in the art to recognize that [he or she] invented what is claimed.” (See Vas-Cath at page 1116.).
Adequate written description requires more than a mere statement that it is part of the invention and reference to a potential method for isolating it. See Fiers v. Revel, 25 USPQ2d 1601, 1606 (CAFC 1993) and Amgen Inc. v. Chugai Pharmaceutical Co. Ltd., 18 USPQ2d 1016.
One cannot describe what one has not conceived. See Fiddles v. Baird, 30 USPQ2d 1481, 1483. In Fiddles v. Baird, claims directed to mammalian FGF’s were found unpatentable due to lack of written description for the broad class. The specification provided only the bovine sequence. Thus, the specification fails to describe these DNA sequences.
For genus claims, an adequate written description of a claimed genus requires more than a generic statement of an invention's boundaries. A patent must set forth either a representative number of species falling within the scope of the genus or structural features common to the members of the genus. Kubin, Exparte, 83 USPQ2d 1410 (Bd. Pat. App. & Int. 2007); Ariad Pharms., Inc. v. Eli Lilly& Co., 598 F.3d 1336, 1350 (Fed. Cir. 2010).
Therefore, only (1) a method for selecting correct pairing of antibody heavy chain (HC) and light chain (LC) in a hetero-IgG bispecific antibody construct, the method comprising:
(a) obtaining a panel of antibody Fab fragments (elected species), wherein each Fab fragment comprises three heavy chain CDRs and three light chain CDRs that specifically bind a first antigen using a high-throughput screening competition chain selective assessment (cCSA) to obtain cognate heavy chain (HC) and light chain (LC) pairing;
(b) obtaining a panel of antibody Fab fragments (species), wherein each Fab fragment and comprises three heavy chain CDRs and three light chain CDRs that specifically bind a second antigen using a high-throughput screening competitive chain selective assessment (cCSA) to obtain cognate heavy chain (HC) and light chain (LC) pairing;
(c) cloning into a vector:
(i) three heavy chain CDRs that specifically bind the first antigen,
(ii) three heavy chain CDRs that specifically bind the second antigen, and
(iii) three light chain CDRs selected from the group consisting of
(a) the three light chain CDRs that specifically bind the first antigen,
(b) the three light chain CDRs that specifically bind the second antigen, and
(c) the three CDRs light chain CDRs that do not specifically bind to either the first antigen or the second antigen;
wherein the vector encodes for a hetero-IgG bispecific antibody construct and a plurality of vectors are generated that encode for a plurality of hetero-IgG bispecific antibody constructs comprising the heavy chain CDRs that bind to each antigen and the light chain CDRs of (c) (iii);
(d) expressing each 4-chain hetero-IgG bispecific antibody construct in a mammalian host cell;
(e) purifying each 4-chain hetero-IgG bispecific antibody construct by Protein A;
(f)(i) measuring the expression levels of each hetero-IgG bispecific antibody construct by A280, and (ii) measuring the binding affinity of each hetero-IgG bispecific antibody construct to the first antigen and the second antigen by Octet, wherein steps (f)(i) and (f)(ii) can be performed simultaneously or in any order; and
(g) comparing the expression levels of (f)(i) and the binding affinities of (f)(ii) for each hetero-IgG bispecific antibody construct by LC-MS in order to identify the optimal pairing of the three heavy chain CDRs that specifically bind a first antigen and the light chain CDRs of (c)(iii) with the optimal pairing of the three heavy chain CDRs that specifically bind a second antigen and the same light chain CDRs of (c)(iii), thereby identify the correct pairing of antibody heavy chain (HC) and light chain (LC) in a hetero-IgG bispecific antibody construct,
(2) said method, wherein the panel of antibody Fab fragments that specifically binds to the first antigen is selected from the group consisting of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine and at least ten antibody Fab fragments,
(3) said method, wherein the panel of antibody Fab fragments that specifically binds to the second antigen is selected from the group consisting of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine and at least ten antibody Fab fragments,
(4) said method wherein the hetero-IgG bispecific antibody construct is selected from the group consisting of Fab/Fab hetero Fc (elected species), scFab/scFab hetero Fc, Fab/scFv hetero Fc, Fab/Fab-scFv hetero Fc, Fab/scFv-Fab hetero Fc, Fab/Fab hetero Fc-scFv, IgG-Fab, scFab-Fc-Fab, IgG-scFv, scFv-IgG, and Fab-scFv-Fc,
(5) Said method wherein the mammalian host cell is a human embryonic kidney cell line HEK293-6E,
(6) a method for selecting correct pairing of antibody heavy chain (HC) and light chain (LC) in a hetero-IgG bispecific antibody construct, the method comprising:
(a) obtaining a plurality of at least two antibody Fab fragments, wherein each Fab fragment comprises three heavy chain CDRs and three light chain CDRs that specifically bind a first antigen using competitive chain selective assessment to obtain cognate heavy chain (HC) and light chain (LC) pairing;
(b) obtaining a plurality of at least two antibody Fab fragments, wherein each Fab fragment comprises three heavy chain CDRs and three light chain CDRs that specifically bind a second antigen using competitive chain selective assessment to obtain cognate heavy chain (HC) and light chain (LC) pairing;
(c) cloning into a vector:
(i) three heavy chain CDRs that specifically bind the first antigen,
(ii) three heavy chain CDRs that specifically bind the second antigen, and
(iii) three light chain CDRs selected from the group consisting of
(a) the three light chain CDRs that specifically bind the first antigen,
(b) the three light chain CDRs that specifically bind the second antigen, and
(c) the three CDRs light chain CDRs that do not specifically bind to either the first antigen or the second antigen;
wherein the vector encodes for a hetero-IgG bispecific antibody construct and a plurality of vectors are generated that encode for a plurality of hetero-IgG bispecific antibody constructs comprising the heavy chain CDRs that bind to each antigen and the light chain CDRs of (c) (iii);
(d) expressing each hetero-IgG bispecific antibody construct in a mammalian host cell, wherein the mammalian host cell is selected from the group consisting of HEK293-6E cells and CHO cells (elected species);
(e) purifying each multispecific antibody construct using Protein A chromatography;
(f)(i) measuring the expression levels of each hetero-IgG bispecific antibody construct using A280 measurement and (ii) measuring the binding affinity of each hetero-IgG bispecific antibody construct to the first antigen and the second antigen using Octet, wherein steps (f)(i) and (f)(ii) can be performed simultaneously or in any order; and
(g) comparing the expression levels of (f)(i) by A280 and the binding affinities of (f)(ii) for each hetero-IgG bispecific antibody construct by Octet in order to identify the optimal pairing of the three heavy chain CDRs that specifically bind to the first antigen and the three light chain CDRs of (c)(iii) with the optimal pairing of the heavy chain CDRs that specifically bind a second antigen and the same light chain CDRs of (c)(iii), thereby obtaining correct pairing of antibody heavy chain (HC) and light chain (LC) in a hetero-IgG bispecific antibody construct,
(7) a method for selecting correct pairing of antibody heavy chain (HC) and light chain (LC) in a hetero-IgG bispecific antibody construct, the method comprising:
(a) obtaining a plurality of antibody Fab fragments, wherein each Fab fragment comprises three heavy chain CDRs and three light chain CDRs that specifically bind a first antigen using competitive chain selective assessment to obtain cognate heavy chain (HC) and light chain (LC) pairing;
(b) obtaining a plurality of antibody Fab fragments, wherein each Fab fragment comprises three heavy chain CDRs and three light chain CDRs that specifically bind a second antigen using competitive chain selective assessment to obtain cognate heavy chain (HC) and light chain (LC) pairing;
(c) cloning the CDRs of the two pluralities into a vector that encodes for a hetero-IgG bispecific antibody construct and a plurality of vectors are generated that encode a plurality of hetero-IgG bispecific antibody constructs comprising the CDRs that bind to each antigen;
(d) expressing each hetero-IgG bispecific antibody (hetero-Fc) combinations in a mammalian host cell;
(e) purifying each secreted hetero-IgG bispecific antibody construct;
(f)(i) measuring the expression levels of each hetero-IgG bispecific antibody construct by A280, and (ii) calculating the percent of correct and incorrect pairing of the HC and the LC species, wherein steps (f)(i) and (f)(ii) can be performed simultaneously or in any order; and
(g) comparing the expression levels of (f)(i) and the percentage of correct and incorrect multispecific antibody construct module species of (f)(ii) for each multispecific antibody construct in order to identify the optimal pairing of three heavy chain CDRs and three light chain CDRs that specifically bind to the first antigen with three heavy chain CDRs and three light chain CDRs that specifically bind the second antigen, thereby obtaining correct pairing of antibody heavy chain (HC) and light chain (LC) in a hetero-IgG bispecific antibody construct, but not the full breadth of the claims meets the written description provision of 35 U.S.C. § 112, first paragraph. Applicant is reminded that Vas-Cath makes clear that the written description provision of 35 U.S.C. § 112 is severable from its enablement provision (see page 1115).
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103(a) which forms the basis for all obviousness rejections set forth in this Office action:
(a) A patent may not be obtained though the invention is not identically disclosed or described as set forth in section 102 of this title, if the differences between the subject matter sought to be patented and the prior art are such that the subject matter as a whole would have been obvious at the time the invention was made to a person having ordinary skill in the art to which said subject matter pertains. Patentability shall not be negatived by the manner in which the invention was made.
The factual inquiries set forth in Graham v. John Deere Co., 383 U.S. 1, 148 USPQ 459 (1966), that are applied for establishing a background for determining obviousness under 35 U.S.C. 103(a) are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims under pre-AIA 35 U.S.C. 103(a), the examiner presumes that the subject matter of the various claims was commonly owned at the time any inventions covered therein were made absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and invention dates of each claim that was not commonly owned at the time a later invention was made in order for the examiner to consider the applicability of pre-AIA 35 U.S.C. 103(c) and potential pre-AIA 35 U.S.C. 102(e), (f) or (g) prior art under pre-AIA 35 U.S.C. 103(a).
Claims 1-3, 5-9, 11-19, 21, 23-27, 29 and 31-35 are rejected under 35 U.S.C. 103 as being unpatentable over Hoogenboom et al (US20060160184, published July 20, 2006; PTO 892) in view of Baeuerle al (US20180134789, published May 17, 2018; PTO 892), Wu et al (US20090215992, published August 27, 2009; PTO 892) and Yin et al (MABS 8(8): 1467-1476, 2016; PTO 892).
Claim 1 recites a method for selecting a multispecific antibody construct, the method comprising:
(a) obtaining a plurality of antibody Fab fragments (elected species), scFvs, or a combination thereof, wherein each Fab fragment and scFv comprises three heavy chain CDRs and three light chain CDRs that specifically bind a first antigen;
(b) obtaining a plurality of antibody Fab fragments (species), scFvs, or a combination thereof, wherein each Fab fragment and scFv comprises three heavy chain CDRs and three light chain CDRs that specifically bind a second antigen;
(c) cloning into a vector:
(i) the CDRs of the three heavy chain CDRs that specifically bind the first antigen,
(ii) the CDRs of the three heavy chain CDRs that specifically bind the second antigen, and
(iii) three light chain CDRs selected from the group consisting of
(a) the CDRs of the three light chain CDRs that specifically bind the first antigen,
(b) the CDRs of the three light chain CDRs that specifically bind the second antigen, and
(c) CDRs of three light chain CDRs that do not specifically bind either the first antigen or the second antigen; wherein the vector(s) encode(s) for one type of multispecific antibody construct module and a plurality of vectors are generated that encode for a plurality of multispecific antibody constructs comprising the heavy chain CDRs that bind to each antigen and the light chain CDRs of (c) (iii);
(d) expressing each multispecific antibody construct in a mammalian host cell;
(e) purifying each multispecific antibody construct;
(f)(i) measuring the expression levels of each multispecific antibody construct and (ii) measuring the binding affinity of each multispecific antibody construct to the first antigen and the second antigen, wherein steps (f)(i) and (f)(ii) can be performed simultaneously or in any order; and
(g) comparing the expression levels of (f)(i) and the binding affinities of (f)(ii) for each multispecific antibody construct in order to identify the optimal pairing of the three heavy chain CDRs that specifically bind a first antigen and the light chain CDRs of (c)(iii) with the optimal pairing of the three heavy chain CDRs that specifically bind a second antigen and the same light chain CDRs of (c)(iii).
Regarding claims 1, 9, 11, 19, 21, 27, 29, 35, Hoogenboom teaches a method for identifying antibodies with desired pairing behavior based on competition selection. Antibodies are selected from a library of antibody fragment, e.g., Fab fragment, see para. [0074] or scFv library, see para. [0169], in particular. The method comprises screening a Fab library that is first enriched on antigen, and the selected heavy chains (each comprises three CDRs) obtained after one or more rounds of selection are then recombined with the selected or unselected light chain repertoire (dashed lines in FIG. 5), and selected again on antigen (FIG. 5, step 4). In this way the selected antibody variable heavy chain domains will have the propensity to bind to the antigen relatively independently of the light chain to which it is paired, see para. [0073]. This method when applied to the isolation of antibodies via the selection of a phage library of Fabs, will yield a high frequency of antibodies that will have an appropriate pairing behavior and high functional yield when produced as mixture by co-expression. The use of competition-selection to bias selected antibodies towards being co-expression compatible, may also be applied to other display libraries (e.g., yeast display libraries), and to in vitro library systems based on ribosome display or mRNA display (Puromycin system), with methods of screening or selection of antibodies that recognize antigen as extensively described in the art, see para. [0074]. The phage Fabs are screened for antigen binding in a binding assay; the pairing behavior between the reactive Fabs and the variable regions of the competing Fab can be further tested by co-expression and binding assays. The preferred format for this selection is the Fab format and not the scFv format, mainly because for most applications whole IgG-type antibodies will need to be established that have interactions between the chains that harbor the variable regions that mimic those seen in the Fab format, see para. [0075]. The method further involves the co-expression of one or more competing antibodies (top, left) in the same host cell as a member of an antibody library (bottom, left). Depicted is the method for Fab fragments, as described in the text. The result of the pairing opportunities of VHCH1 (white boxes) chain when co-expressed with two other Fab fragments is depicted. The original combination of the VH with its cognate light chain (hatched box), will retain its original binding affinity for antigen and can thus be selected, see para. [0032], FIG. 6, para. [0043], Examples 4, 5, 6, 7, in particular.
Hoogenboom teaches that antibodies with pairing-compatible variable region sequences and, therefore, suitable pairing behavior of variable regions, are identified by a variety of methods that are disclosed within this document. In a first approach, antibodies with pairing-compatible variable regions are selected from panels of antigen-specific antibodies (in which the antigen can be one defined target antigen but also a collection of different antigens, and the panel contains at least two antibodies), as follows. The sequences of heavy and light variable regions are determined and inspected to find clones with identical or highly similar light or heavy chain variable domains. If the amino acid sequence of part of or the complete variable region is identical for two antibodies, the two given antibodies have a pairing-compatible variable region, see para. [0064]. The mixture may contain a given selection of antibodies, recognizing epitopes on the same or different targets, see para. [0133], [0134], in particular. Suitable target antigens for antibody mixtures in oncological diseases are many, including CD19, CD20, CD22, CD25 (IL-2 receptor), CD33, the IL-4 receptor, EGF-receptor, mutant EGF receptor, Carcino-Embryonic Antigen, Prostate-specific Antigen, ErbB2IHER2, Lewis.sup.y carbohydrate, Mesothelin, Mucin-1, the transferrin receptor, Prostate-specificMembrane Antigen, VEGF and receptors, EpCAM and CTLA-4, see para. [0141].
Hoogenboom teaches that recombinant DNA technology provides methods well known in the art to clone the variable region genes, and the binders are subcloned to produce the bispecific antibody in cell lines, see para. [0127]. Typically, expression vectors used in the host cells will contain sequences for plasmid maintenance and for cloning and expression of exogenous nucleotide sequences.
Hoogenboom teaches that a recombinant expression vectors encoding at least one antibody heavy or light chain is introduced into a mammalian cells, e.g., CHO cells. In many instances the expression vector may contain both heavy and light chain genes, and co-transfection will lead to the production of intact antibody, recovered from the culture medium, see para. [0112], [0121], in particular. Expression of three Fab fragments in the same eukaryotic cell, see Example 13. Antibodies with the same light chain are isolated against three different antigen, TNF-alpha, interleukine-1beta (IL1-beta) and interleukin-6 (IL-6), see Example 14, in particular. In vitro pairing of antibody chains produced in different cells to form defined antibody mixtures, see Example 15, in particular.
Claims 2, 3, 12, 13 are included because it is within the purview of one of ordinary skilled in the art to co-express or produce any number of Fab such as two that bind to a first antigen and any number of Fab, such as two that bind to a second antigen in a host cell using recombinant technology as taught by Hoogenboom.
Regarding claims 5, 15, 23, 27, 31, 35, Hoogenboom teaches that mammalian host cells include Chinese Hamster Ovary (CHO cells), NS0 myeloma cells and SP2 cells, C127, 3T3, CHO, human epidermal A431 cells, Jurkat, U937, HL-60, mouse L-cells, Baby Hamster Kidney cells, COS or CV-1 cells, PER.C6 cells, see para. [0116].
Regarding claims 8, 18, 26, 27, 34, 35, Hoogenboom teaches that the multispecific antibody can be purified by affinity chromatography with protein A, protein G and other affinity matrices, see para. [0093], [0112].
Hoogenboom teaches that optimal pairing of heavy and light chain variable regions (para. [0099], [0101]) can be identified by expression levels (para. [0100]) and binding affinity, para. [0018], [0032], [0057], [0065], [0072].
Regarding claims 7, 9, 21, 25, 27, Hoogenboom teaches that binding affinity can be Surface Plasmon Resonance (SPR) analysis, see para. [0080].
Hoogenboom does not teach the method wherein the expression levels are determined by a method of A280 measurement SDS-PAGE as per claims 6, 16, 19, 21, 24, and 32, or wherein the affinity of each multispecific antibody construct to the first and the second antigen is measured using Octet, SPR or flow cytometry as per claims 7, 9, 21, 25, 27 and wherein the method uses liquid chromatography-mass spectrometry (LC-MS) to determine the percentage of correct and incorrect multispecific antibody construct species as per claims 17, 29, 33, and 34.
However, Baeuerle teaches bispecific antibody, e.g., scFv-scFv that binds to CD3ε and any tumor antigen, e.g., EGFR; protein level of antibody was determined by A280 and expression levels were back-calculated. The expected molecular weight was determined on an SDS-PAGE as per claims 6, 16, 19, 21, 24, and 32, see para. [0360].
Regarding claims 7, 9, 21, 25, 27, Baeuerle teaches that antibody binding affinity to CD3 can be determined, for example, by Surface Plasmon Resonance (SPR), see para. [0277] or Octet, see para. [0119], [0120], [0345], [0346].
Likewise, Wu teaches the levels of bispecific dual variable domain IgG (DVD-Ig) can be determined by various method, e.g., SDS-PAGE and A280 or bicinchronic acid (BCA), see para. [0328].
Regarding claims 7, 9, 21, 25, 27, Wu teaches that the binding affinity of DVD-Ig antibody can be determined by surface plasmon resonance (SPR), see para. [0334].
Hoogenboom, Baeuerle and Wu do not teach the uses of liquid chromatography-mass spectrometry (LC-MS) to determine the percentage of correct and incorrect multispecific antibody construct species as per claims 17, 29, 33, and 34.
However, Yin teaches the use of liquid chromatography-mass spectrometry (LC-MS) to analyze the protein A-purified BsIgG to determine the percentage of correct and incorrect pairing between heavy and common light chain of various bispecific IgG antibodies (BsIgG), see entire document, p. 1468, Detection of IgG using LC-MS, in particular. The observed IgG species included the correctly paired anti-HER2/CD3 BsIgG (H1L1/H2L2) and the isobaric light chain-scrambled IgG (H1L2/H2L1). The additional IgG species contained 2 copies of either the anti-HER2 light chain (H1L1/H2L1) or the anti-CD3 light chain (H1L2/H2L2). The mass difference between the BsIgG and either of the IgG containing 2 copies of L1 or L2 reflects the mass difference between the 2 light chains. Yin teaches that the intrinsic cognate heavy and light chain pairing preference was observed for anti-VEGFA and anti-VEGFC BsIgG antibodies, resulting in 68.5% yield of BsIgG. In contrast, the heavy and light chains of anti-HER2 and anti-CD3 antibodies paired randomly, giving rise to 24.7% anti-HER2/CD3 BsIgG (Table S4 and S5). Before mathematical correction, these estimates were 70.6% BsIgG for anti-VEGFA/VEGFC and 49.4% BsIgG for anti-HER2/CD3, reflecting a deceptively high estimate of properly paired BsIgG in the latter case. The quantification platform developed here provides a potentially broadly applicable tool to detect and quantify any cognate chain preference when pairs of antibodies are co-expressed. The quantification platform developed here offers high reproducibility and robustness, allowing for the rapid analysis of hundreds of clones in an automated fashion. Applications extend beyond evaluating BsIgG yields of different designs, such as screening clones in the development of stable cell lines. Thus, the platform has the potential to be broadly useful in the development of BsIgG therapeutics, see p. 1473, right col.. This Orbitrap-based high resolution LC-MS platform performance is superior to the QTOF-based LC-MS system due to improved desolvation and increased signal-to-noise ratio.
In view of the combined teachings of the references, it would have been prima facie obvious to a person of ordinary skill in the art before the effective filling date of the claimed invention to have produced the method by combining the method of identifying optimal pairing between heavy chain comprising 3 CDRs and light chain comprising 3 CDRs in candidate Fab or scFv sequences by high-throughput competition screening for correct HC/LC pairing to identify common light chain, expressing each bispecific antibody construct in a mammalian host cell such as CHO cells, purify the expressed antibody, typically using protein A affinity chromatography, measure the expression levels and binding affinity of the bispecific antibody of Hoogenboom wherein the protein levels are determined by any method known in the art, e.g., A280 and SDS-PAGE as taught by Baeuerle or Wu and wherein the binding affinity is determined by any method known in the art, e.g., Surface Plasmon Resonance (SPR) or Octet as taught by Baeuerle in in order to identify the best pairing of HC/LC for bispecific antibody and then using high resolution LC-MS of Yin to calculate the percentage of correct and incorrect species in the BsIgG antibody. It is within the purview of one of ordinary skill in the art to compare the expression level and binding affinity for each bispecific antibody in order to identify the optimal pairing between antibody heavy and light chains or common light chains wherein each heavy chain comprising three CDRs and each light chain comprising three CDRs as taught by Hoogenboom.
One of ordinary skill in the art would have had a reasonable expectation of success in using competitive and non-competitive screening methodology to identify cognate heavy and light chain binding pairs in order to improve the correct heavy chain-light chain pairing of IgG bispecific antibody. The use of one common light chain avoids the formation of heterodimers in which pairing of light and heavy chains results in antigen binding domains that are notfunctional.
One of ordinary skill in the art would have been motivated to do so because Hoogenboom teaches that the method identified antibodies that exhibit a native preference toward cognate heavy and light chain pairing, that enables the production of hetero IgG multispecific antibody without further engineering, see para. [0077], [0186]. The IgG-like bispecific antibodies format has favorable properties due to their resemblance to conventional IgG , e.g., stability and solubility compared to scFv; the Fc maintains effector function, e.g., ADCC and CDC.
One of ordinary skill in the art would have had a reasonable expectation of success, because measuring protein expression levels is typically determined using A280 measurement and reduce SDS-PAGE or BCA as evidenced by Wu, see para. [0328] and used Surface Plasmon Resonance (SPR), or Octet is commonly used for determining antigen-antibody binding affinity as evidenced by Baeuerle, see para. [0277], [0119], [0120], [0345], [0346].
One of ordinary skill in the art would have been motivated to do so because Yin teaches that LC-MS method is a fast based on chromatographic peak identification and mass to assess mispaired species; the high resolution, LC-MS quantification platform offers high reproducibility and robustness, allowing for the rapid analysis of hundreds of clones in an automated fashion and has the potential to be broadly useful in the development of BsIgG therapeutics, see p. 1473, in particular.
In addition, the claims would have been obvious because "a person of ordinary skill has good reason to pursue the known options within his or her technical grasp. If this leads to the anticipated success, it is likely the product not of innovation but of ordinary skill and common sense". See KSR International Co. v. Teleflex Inc., 82 USPQ2d 1385 (U.S. 2007).
“The test of obviousness is not express suggestion of the cl aimed invention in any or all of the references but rather what the references taken collectively would suggest to those of ordinary skill in the art presumed to be familiar with them.” See In re Rosselet 146 USPQ 183, 186 (CCPA 1965).
“There is no requirement (under 35 USC 103(a)) that the prior art contain an express suggestion to combine known elements to achieve the claimed invention. Rather, the suggestion to combine may come from the prior art, as filtered through the knowledge of one skilled in the art.,” Motorola, Inc, v. Interdigital Tech. Corn., 43 USPQ2d 1481, 1489 (Fed. Cir. 1997).
Accordingly, the claimed invention as a whole was prima facie obvious to one of ordinary skill in the art before the effective filling date of the claimed invention especially in the absence of evidence to the contrary.
Claims 4, 10, 14, 20, 22, 28, 30 and 36 are rejected under 35 U.S.C. 103 as being unpatentable over Hoogenboom et al (US20060160184, published July 20, 2006; PTO 892) in view of Baeuerle al (US20180134789, published May 17, 2018; PTO 892), Wu et al (US20090215992, published August 27, 2009; PTO 892) and Yin et al (MABS 8(8): 1467-1476, 2016; PTO 892) as applied to claims 1-3, 5-9, 11-19, 21, 23-27, 29 and 31-35 mentioned above and further in view of Kannan et al (WO2009089004, published July 16, 2009; PTO 892).
The combine teachings of Hoogenboom, Baeuerle and Wu have been discussed supra.
The reference does not teach the method wherein the multispecific construct module include at least two modules of Fab/Fab hetero Fc or scFv-IgG as per claims 4, 10, 14, 20, 22, 28, 30 and 36.
However, Kannan teaches a method of making bispecific (BsAbs) antibody Fc heterodimer using electrostatic steering effect to promote dimerization between Fc from two different IgGs antibodies by substituting one or more residues that make up the CH3-CH3 interface in both domains with a charged amino acid such that homodimer is electrostatically unfavorable but heterodimerization is electrostatically favorable, see entire document, Summary of invention, reference claims 1-34, in particular. The antibody is a bispecific IgG antibody comprises two different Fab (aka Fab/Fab hetero Fc) or two scFvs fused to a hetero Fc (aka scFv-IgG) having different binding specificity for two or more antigens, see Fig. 2, p. 5, caption of Fig. 2, in particular.
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Kannan teaches that the IgG Fc is a human IgG1 Fc, see reference claim 40-41, in particular. The first CH3 containing Fc comprises a replacement of Lys409 with a negative-charged amino acid and the second CH3-containing Fc comprises a replacement of Asp399, Asp356 or Glu357 with a positive-charged amino acid to promote heterodimer formation, see p. 3, reference claims 60-68, in particular. Kannan further teaches Fc heterodimer encompasses “knobs-into-holes”, see p. 2, line 5-8. Kannan teaches that the Fc may contain additional alteration, such as knobs-into-holes mutations, see p. 3, line 29-30 or combined with inter-CH3 domain disulfide bond to enhance heterodimer formation, see p. 2, line 17-20. The enhanced heterodimerization will maximize the yield of bispecific IgG antibodies, see p. 2, line 19-20, in particular.
In view of the combined teachings of the references, it would have been prima facie obvious to a person of ordinary skill in the art before the effective filling date of the claimed invention to have produced the claimed method by combining the method of Hoogenboom, Baeuerle and Wu with Kannan’s electrostatically to facilitate pairing between two Fcs from two different IgG antibodies with a reasonable expectation of success, e.g., electrostatically favorable heterodimerization.
One of ordinary skill in the art would have been motivated to do so because Kannan teaches that the desired heterodimerization is maximized due to electrostatic attraction between the Fc of two heavy chains, and improved the yield of bispecific IgG antibodies, see p. 2, line 19-20, in particular.
In addition, the claims would have been obvious because "a person of ordinary skill has good reason to pursue the known options within his or her technical grasp. If this leads to the anticipated success, it is likely the product not of innovation but of ordinary skill and common sense". See KSR International Co. v. Teleflex Inc., 82 USPQ2d 1385 (U.S. 2007).
“The test of obviousness is not express suggestion of the cl aimed invention in any or all of the references but rather what the references taken collectively would suggest to those of ordinary skill in the art presumed to be familiar with them.” See In re Rosselet 146 USPQ 183, 186 (CCPA 1965).
“There is no requirement (under 35 USC 103(a)) that the prior art contain an express suggestion to combine known elements to achieve the claimed invention. Rather, the suggestion to combine may come from the prior art, as filtered through the knowledge of one skilled in the art.,” Motorola, Inc, v. Interdigital Tech. Corn., 43 USPQ2d 1481, 1489 (Fed. Cir. 1997).
Accordingly, the claimed invention as a whole was prima facie obvious to one of ordinary skill in the art before the effective filling date of the claimed invention especially in the absence of evidence to the contrary.
Conclusion
No claim is allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to PHUONG HUYNH whose telephone number is (571)272-0846. The examiner can normally be reached on 9:00 a.m. to 6:30 p.m. The examiner can also be reached on alternate alternative Friday from 9:00 a.m. to 5:30 p.m.
If attempts to reach the examiner by telephone are unsuccessful, the examiner's supervisor, Misook Yu, can be reached at 571-272-0839. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/PHUONG HUYNH/ Primary Examiner, Art Unit 1641
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