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 .
DETAILED ACTION
Claims 1-14 and 17-23 are pending in the instant application and being examined on the merit.
Priority
Applicant’s claim for the benefit of a prior-filed application under 35 U.S.C. 119(e)
or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged. Applicant has not
complied with one or more conditions for receiving the benefit of an earlier filing date
under 35 U.S.C. 119(e) as follows:
The later-filed application must be an application for a patent for an invention
which is also disclosed in the prior application (the parent or original nonprovisional
application or provisional application). The disclosure of the invention in the parent
application and in the later-filed application must be sufficient to comply with the
requirements of 35 U.S.C. 112(a) or the first paragraph of pre-AIA 35 U.S.C. 112,
except for the best mode requirement. See Transco Products, Inc. v. Performance
Contracting, Inc., 38 F.3d 551, 32 USPQ2d 1077 (Fed. Cir. 1994).
The disclosure of the prior-filed priority applications, EP21173577.4 and
PCT/EP2022/062868, fail to provide adequate support or enablement in the manner
provided by 35 U.S.C. 112(a) or pre-AIA 35 U.S.C. 112, first paragraph for one or more
claims of this application.
Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55
for EP21173577.4 and PCT/EP2022/062868. Claim analysis of previous priority applications reveal the effective filing dates of claims are drawn to two separate applications, namely, PCT/EP2022/062868 (filed 05/12/2022) and the original foreign priority application EP21173577.4 (filed 05/12/2021). The priority application EP21173577.4 only describes the humanized antibodies 2-5D3, 5-5D3, and 6-5D3 and
specifically lacks priority for the humanized 5D3-6 variants LVg2, HVg2, HVg3, HVg4, HVg5, HVg7, LVg2HVg2, LVg2HVg3, LVg2HVg4, LVg2HVg5, and LVg2HVg7 which were described for the first time in PCT/EP2022/062868 in Table 4 on pages 34-35. Thus, changes to the CDR region of an antibody that binds PSMA comprising a VH comprising SEQ ID NO:30 and a VL comprising SEQ ID NO:31 were first introduced in PCT/EP2022/062868. Thus, claim 1 and its dependents, claims 2-14 and 17-23 have a priority date of 05/12/2022.
Objections to the Claims
Claim 22 is objected to because of the following informalities:
Regarding instant claim 22, the subject “a tumor” should be before “in vivo” in line 1 as ---a tumor in vivo--- .
Appropriate correction is required.
Claim Rejections – 35 USC § 112(b)
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 1-14 and 17-23 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 the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Regarding instant claim 1, claim 1 recites that for a specific group of X variables, at least one applies, but it does not identify the selectable options if only one applies. It is unclear if when only one X variable is selected for the group if the other X can be deleted or be any other amino acid. Thus, the claim is indefinite. The X groups are present in:
Page 4, line 7 and page 5, line 14 of claim 1, for (X1) to (X3) at least one applies;
Page 4, line 15 and page 5, line 23 of claim 1, for (X5) and (X6) at least one applies;
Page 4, line 19 and page 5, line 27 of claim 1, for (X7) and (X8) at least one applies;
Page 4, line 23 and page 6, line 2 of claim 1, for (X9) and (X10) at least one applies; and
Page 5, line 6 and page 6, line 9 of claim 1, for (X11) and (X12) at least one applies.
Claims 2-14 and 17-23 also contain the unclear subject matter and are also rejected.
Claim Rejections – 35 USC § 112(a)
Claims 1-14 and 17-23 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 applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
Regarding instant claim 1, an antibody that binds PSMA which comprises a VH comprising SEQ ID NO:30 and a VL comprising SEQ ID NO:31, wherein within the VH and VL-CDR regions the X1-X12 variables are selectable from a series of amino acids is claimed, but the disclosure does not test: a) any mutations of CDR residues of VH X2-X4 and X8-X9 (amino acid positions 31-32, 34, 58 and 61) or VL X12 (amino acid position 31); and b) only tests 12 h5D3 clones, namely the humanized 5D3-6, and its variants LVg2, HVg2, HVg3, HVg4, HVg5, HVg7, LVg2HVg2, LVg2HVg3, LVg2HVg4, LVg2HVg5, and LVg2HVg7. The 12 h5D3 clones tested do not encompass all amino acids claimed as substitutes within X1-X12 and do not encompass are variation to allow mixing and matching of separate clone mutations. Claims 2-14 and 17-23 are dependent on claim 1 and also allow for variation of the CDR and are rejected.
Regarding claim 13, a method for producing an antibody is claimed comprising culturing a non-human host, but the specification only describes culturing ---non-human host cells--- ;
Claims 22-23 are for methods of treating, inhibiting, or diagnosing a tumor in vivo comprising: i) a PSMA antibody defined above; ii) a nucleic acid composition encoding the antibody above; or iii) a non-human host comprising a nucleic acid that encodes the antibody above, but the instant specification does not have examples of successfully: A) treating or inhibiting a tumor in vivo; or B) diagnosing a tumor in vivo: a) that is “used” in vivo rather than ---administered---; b) that does not express PSMA; or c) that is administered in a non-human host rather than a plasmid or antibody.
Scope of the claimed genus
Claim 1 claims an antibody that binds PSMA which comprises a VH comprising SEQ ID NO:30 and a VL comprising SEQ ID NO:31, wherein within the VH and VL-CDR regions the X1-X12 variables are selectable from a series of amino acids. Claims 2-14 and 17-23 are dependent on claim 1 and also allow for variation of the CDR and are rejected.
Claim 13 claims a method for producing an antibody comprising culturing a non-human host comprising a nucleic acid encoding the antibody above under conditions to allow synthesis of the antibody;
Claims 22-23 are for methods of treating, inhibiting, or diagnosing a tumor in vivo comprising: i) a PSMA antibody defined above; ii) a nucleic acid composition encoding the antibody above; or iii) a non-human host comprising a nucleic acid that encodes the antibody above
Summary of Species disclosed in the original specification
MPEP § 2163 states that a “representative number of species” means that the species which are adequately described are representative of the entire genus. Thus, when there is substantial variation within the genus, one must describe a sufficient variety of species to reflect the variation within the genus.
1) The murine antibody 5D3 was known to the prior art (US 2019/ 0330367 Barinka C et al.), wherein Barinka taught the 5D3 antibody can effectively bind PSMA protein and PSMA expressed on tumor cells (Fig. 11 and 13). In the instant application the murine antibody 5D3 was humanized and back mutations were introduced into each model based on a visual inspection of the aligned structures of the 5D3 variable domains and search hits in order to avoid local structural incompatibilities between CDRs of the mouse donor antibody and the human acceptor framework regions (specification, page 30 last paragraph to page 31 first paragraph). Humanization of 5D3 produced three separate antibodies, namely 2-5D3, 5-5D3, and 6-5D3 wherein amino acid grafting of the CDRs of 5D3 and humanization produced antibodies with variable properties ( specification page 33, Table 2).
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217
736
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Alignment of Kabat defined VH and VL CDRs with Clustal Omega showed that the CDRs were the same.
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114
672
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While the CDR was maintained: 1) 2-5D3 and 5-5D3 had diminished yield (specification page 33, Table 2); 2-5D3 had stability issues (Fig. 9); and 3) 5-5D3 lacked specificity (Fig. 11). Only 6-5D3 was able to maintain acceptable yield, stability, and specificity.
The instant specification taught three humanized variants of a 5D3 murine antibody, wherein the 6-5D3-lgG1 (6-5D3-hFab) performs well as it is the only one that retained high expression yields, temperature stability and specificity of the parent 5D3 mAb. It was unexpected that a humanized variant of 5D3 murine antibody can be obtained which retains these three favorable characteristics of the parent 5D3 mAb despite the CDR-grafting during humanization (page 33, second paragraph).
Alignment of 5D3 and humanized 6-5D3 which includes back mutations show about an 73% identical match with the VH of 5D3 and an 88% match with the VL of 5D3. Mutations in 6-5D3 further fall within the Vernier Zone that include VH G49S, L69T, and A78L.
VH
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239
631
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VL
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237
626
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Further, in silico humanization of the 5D3 antibody using BioPhi an in silico antibody humanization tool available after the priority date was unable to identify the humanization sequence claimed (BioPhi et al. https://biophi.dichlab.org/humanization/humanize/ 2026).
VH 5D3-6 vs humanized in silico
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227
619
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628
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The instant specification taught generation of 6-5D3 variant clones LVg2, HVg2, HVg3, HVg4, HVg5, HVg7, LVg2HVg2, LVg2HVg3, LVg2HVg4, LVg2HVg5, and LVg2HVg7, wherein site-directed mutagenesis was performed at different locations throughout the VH and VL CDR. Table 4 identifies the locations of amino acid mutations tested as VH 30, 55, 56, 57, 62 and VL 31. In claim 1, the locations of the tested antibody mutants in a VH comprising SEQ ID NO:30 and a VL comprising SEQ ID NO: 31 are indicated as X1, X5-X7, and X10-X11. No mutations were tested at VH 31-32, 34, 58, 61, and VL 31 indicated as X2-X4, X8-X9, and X12 in a VH comprising SEQ ID NO:30 and a VL comprising SEQ ID NO: 31 in claim 1.
Further, only a small subset of amino acids were exchanged in comparison to the amino acids claimed in claim 1. See table below.
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200
400
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The Applicant has written description of 5D3-6 and its variants LVg2, HVg2, HVg3, HVg4, HVg5, HVg7, LVg2HVg2, LVg2HVg3, LVg2HVg4, LVg2HVg5, and LVg2HVg7 for individual antibody clones with the following X1-X12, wherein the amino acids from separate mutants are not mixed between clones.
5D3-6 N, T, S, M, D, G, D, T, N, G, N, N
LVg2 N, T, S, M, D, G, D, T, N, G,E, N
HVg2 Q, T, S, M, D, A, D, T, N, A, N, N
HVg3 Q, T, S, M, E, G, E, T, N, A, N, N
HVg4 Q, T, S, M, E, G, D, T, N, A, N, N
HVg5 Q, T, S, M, E, G, S, T, N, A, N, N
HVg7 Q, T, S, M, D, G, D, T, N, G, N, N
LVg2HVg2 Q, T, S, M, D, A, D, T, N, A, E, N
LVg2HVg3 Q, T, S, M, E, G, E, T, N, A, E, N
LVg2HVg4 Q, T, S, M, E, G, D, T, N, A, E, N
LVg2HVg5 Q, T, S, M, E, G, S, T, N, A, E, N
LVg2HVg7 Q, T, S, M, D, G, D, T, N, G, E, N
Alterations of CDR residues were shown in Table 4 to alter antibody binding. It is unknown if antibody yield and specificity were altered as shown in other humanized antibodies above.
Mixture of CDR residues that were tested could potentially have very large effects as seen when comparing HVg5 and LVg2HVg3, wherein changing 2 of the CDR residues show about a 50-fold change in antibody affinity
Clone X1-X10 ELISA (nM)
HVg5 Q, T, S, M, E, G, S, T, N, A, N, N 0.06
LVg2HVg3 Q, T, S, M, E, G, E, T, N, A, E, N 2.69
As noted below, the art generally accepted that the combination of the CDRs within the VH and VL pair of an antibody were essential for binding specificity. The specification does not describe what residues within the CDRs confer the binding activity claimed without altering antibody secretion, stability, or specificity and the claim language permits changes in the VH and VL that contain the CDRs in the VH or VL. Accordingly, the skilled artisan would not be able to discern a structure/function correlation for antibodies other than those comprising all six CDRs of a PSMA antibody of 5D3-6, and its variants LVg2, HVg2, HVg3, HVg4, HVg5, HVg7, LVg2HVg2, LVg2HVg3, LVg2HVg4, LVg2HVg5, and LVg2HVg7.
The instant specification does not teach methods for culturing a non-human host comprising a nucleic acid molecule that encodes an antibody that is not a host cell.
3) The instant specification taught 5D3-6, and its variants LVg2, HVg2, HVg3, HVg4, HVg5, HVg7, LVg2HVg2, LVg2HVg3, LVg2HVg4, LVg2HVg5, and LVg2HVg7y effectively bind PSMA in Fig. 10 and 16. The instant specification does not have examples of successfully: A) treating or inhibiting a tumor in vivo; or B) diagnosing a tumor in vivo: a) that is “used” rather than ---administered---; b) that does not express PSMA; or c) administering a non-human host.
State of the Relevant Art
1) The murine antibody 5D3 was known to the prior art (US 2019/0330367 Barinka C et al.), wherein Barinka taught the 5D3 antibody can effectively bind PSMA protein and PSMA expressed on tumor cells (Fig. 11 and 13). A structure activity relationship that would permit alterations of the 5D3 antibody VH and VL CDR was not known wherein binding, yield, stability, and antibody secretion were optimized was not known to the prior art.
The prior art taught CDR grafting may result in reduced target binding even if the VH–VL interface residues are preserved (Chiu ML et al. (Antibodies 2019, 8(4), 55), page 16, 2.1.4 Back Mutations section, paragraph 1). Chiu taught any CDR grafting protocol must include a step to identify FR positions that are critical for maintaining the CDR conformation (page 16, 2.1.4 Back Mutations section, paragraph 1 ). Chiu taught if a non-human residue in a critical position cannot be preserved because there are no such human sequences, one usually applies the so-called back mutation, i.e., a mutation of a residue in the human FR to the amino acid that occurs in the non-human parent (page 16, 2.1.4 Back Mutations section, paragraph 1). Chiu taught back mutations reduce the humanness score of the resulting variant, but the change should improve the binding affinity (page 16, 2.1.4 Back Mutations section, paragraph 1) and may further improve expression of the antibody variants (page 17, paragraph 3). Chiu taught the central zone around CDR-H3 remains problematic for accurate modeling and the lack of information on the CDR involvement in antigen binding often leads to an excessive number of back mutations in the humanized antibodies (page 17, paragraph 2). Chiu taught to avoid such outcomes, each potentially critical position should be tested for back mutation and only those mutations that affect binding should be incorporated into the final antibody (page 17, paragraph 2). Thus, individual back mutations affect target binding, expression, and humanness of the antibody.
Regarding the location of the back mutations, Chiu taught back mutations in the VH-VL interface and that are in direct contact with CDRs, or “Vernier zone” (Chiu Fig 7-8). The Vernier zone constitutes 30 residues, wherein 4 are considered part of HCDR1 in the Chothia and IMGT definitions and the remaining are divided between the VH and VL (page 16, 2.1.4 Back Mutations section, paragraph 2). Chiu taught a careful analysis of the importance of each Vernier zone position in the context of given CDRs and antibody–antigen interactions is the cornerstone of the humanization process (page 16-17, bridging sentence). Thus, individual back mutations at specific positions within a humanized antibody affect target binding, expression, and humanness of the antibody and individual mutations are required for generation of an effective antibody.
At the time of the filing of the instant application, it was well established in the art that the formation of an intact antigen-binding site in an antibody usually required the association of the complete heavy and light chain variable regions of a given antibody, each of which consists of three “complementarity determining regions” (“CDRs”) which provide the majority of the contact residues for the binding of the antibody to its target epitope. E.g., Almagro & Fransson, Frontiers in Bioscience 2008; 13:1619-33; (see Section 3 “Antibody Structure and the Antigen Binding Site” and Figure 1). While affinity maturation techniques can result in differences in the CDRs of the antibody compared to its parental antibody (page 3 “The IgG Molecule, second and third paragraphs), those techniques involve trial-and-error testing and the changes that maintain or improve affinity are not predictable a priori. E.g., id., (page 6 ending paragraph onto page 7). Chiu ML et al. (Antibodies 2019 8, 55, 1-80) taught the antigen binding of antibodies often results in conformational changes in the contact surface areas of both the antibody and the antigen (page 5, first paragraph). Chiu further taught antibody modeling has been shown to be accurate for the framework region sequences, but CDR modeling requires further development and improvements (page 6, second paragraph).
In addition to changes within the CDR altering target binding, alterations to the CDR have been shown to dramatically alter antibody secretion. Hasegawa H et al. (mAbs 2017, 9(5) 854-873) taught a pair of human IgG clones with a single amino acid substitution in the variable region was sufficient to alter the efficiency of immunoglobulin biosynthesis (page 866, last sentence left column). Hasegawa taught the 2 mAbs differed only by one amino acid in the LC's CDR1 and that despite the near-identity of their primary sequences, the parental mAb secreted copious amounts of IgG to the culture media, while the variant mAb induced RB phenotypes extensively and secreted 20-fold less IgG (page 866, right column, first paragraph). Importantly, the 2 model IgGs were by no means abnormal or defective as mAbs, but demonstrated a profound impact of a single amino acid substitution on immunoglobulin biosynthesis (page 866, right column, first paragraph).
2) The prior art taught production of a 5D3 antibody that targeted PSMA in the non-human host cells E. coli (Zora Novakova Z et al. (Int J Mol Sci. 2020 Sep 12;21(18):6672) IDS reference page 13 Section 4.9), but the prior art does not teach a method of producing an antibody by culturing non-human hosts that are not cells.
3) The murine antibody 5D3 was known to the prior art (US 2019/0330367 Barinka C et al.), wherein Barinka taught the 5D3 antibody can effectively bind PSMA protein and PSMA expressed on tumor cells (Fig. 11 and 13). Banerjee et al., (Journal of Nuclear Medicine 2019 60.3: 400-406 IDS reference) taught 111In-DOTA-5D3 is a radiolabeled antibody for imaging and a surrogate for therapy of malignant tissues expressing PSMA (abstract). Huang C et al. (Mol Pharm. 2020 Aug 17;17(9):3392–3402) taught anti-PSMA monoclonal antibodies (mAbs) have been developed as bioligands for diagnostic imaging and targeted PC therapy (abstract). Huang taught administration of an antibody drug conjugate of 5D3-DM1 to a subject with PSMA expressing cancer could effectively decrease tumor growth (abstract). The parental 5D3 antibody is not known to effectively treat subjects with cancer without a toxin conjugated to the antibody. The instant humanized 5D3-6 and variant antibodies in the instant specification would not be expected to effectively treat PSMA expressing tumors without being conjugated to a toxic agent.
Administration of antibodies and plasmids that encode antibodies is known to the prior art. Kim H et al. (Cancer Gene Therapy 2016 23, 341–347) taught administration of antibodies or plasmids encoding antibodies as an effective method to cause antibodies to enter the bloodstream in vivo (abstract). The prior art does not show a method of effectively administering a non-human host in vivo for diagnostic purposes.
The prior art has not described using a PSMA targeted antibody to diagnose a tumor that does not express PSMA.
Claims 1-16 and 18-30 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for:
1) an antibody that binds PSMA comprising 5D3-6, and its variants LVg2, HVg2, HVg3, HVg4, HVg5, HVg7, LVg2HVg2, LVg2HVg3, LVg2HVg4, LVg2HVg5, and LVg2HVg7y;
2) a method of diagnosing a tumor that expresses PSMA in vivo comprising administering to a subject in need thereof the antibody 5D3-6, or its variants LVg2, HVg2, HVg3, HVg4, HVg5, HVg7, LVg2HVg2, LVg2HVg3, LVg2HVg4, LVg2HVg5, and LVg2HVg7y or a plasmid comprising the antibodies above;
does not reasonably provide enablement for:
an antibody that binds PSMA comprising a VH of SEQ ID NO:30 and a VL of SEQ ID NO:31 with variable X1-X12 that are not 5D3-6, and its variants LVg2, HVg2, HVg3, HVg4, HVg5, HVg7, LVg2HVg2, LVg2HVg3, LVg2HVg4, LVg2HVg5, and LVg2HVg7y
2a) a method of treating or inhibiting a tumor in vivo by administering to a subject in need thereof the antibody above;
2b) a method of diagnosing a tumor in vivo with the antibody above, wherein the tumor does not express PSMA, and wherein a non-human host cell comprising the antibody is administered to the subject in need thereof;
The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make the invention commensurate in scope with these claims.
Regarding instant claim 1, an antibody that binds PSMA which comprises a VH comprising SEQ ID NO:30 and a VL comprising SEQ ID NO:31, wherein within the VH and VL-CDR regions the X1-X12 variables are selectable from a series of amino acids is claimed, but the disclosure does not test: a) any mutations of CDR residues of VH X2-X4 and X8-X9 (amino acid positions 31-32, 34, 58 and 61) or VL X12 (amino acid position 31); and b) only tests 12 h5D3 clones, namely the humanized 5D3-6, and its variants LVg2, HVg2, HVg3, HVg4, HVg5, HVg7, LVg2HVg2, LVg2HVg3, LVg2HVg4, LVg2HVg5, and LVg2HVg7. The 12 h5D3 clones tested do not encompass all amino acids claimed as substitutes within X1-X12 and do not encompass are variation to allow mixing and matching of separate clone mutations. Claims 2-14 and 17-23 are dependent on claim 1 and also allow for variation of the CDR and are rejected.
Claims 22-23 are for methods of treating, inhibiting, or diagnosing a tumor in vivo comprising: i) a PSMA antibody defined above; ii) a nucleic acid composition encoding the antibody above; or iii) a non-human host comprising a nucleic acid that encodes the antibody above, but the instant specification and the prior art do not have examples of successfully administering the antibody for: A) treating or inhibiting a tumor in vivo; or B) diagnosing a tumor in vivo: a) that is “used” in vivo rather than ---administered---; b) that does not express PSMA; or c) that is administered in a non-human host rather than a plasmid or antibody.
There are many factors to be considered when determining whether there is sufficient evidence to support a determination that a disclosure does not satisfy the enablement requirement and whether any necessary experimentation is "undue." These
factors include, but are not limited to:
(A) The breadth of the claims;
(B) The nature of the invention;
(C) The state of the prior art;
(D) The level of one of ordinary skill;
(E) The level of predictability in the art;
(F) The amount of direction provided by the inventor;
(G) The existence of working examples; and
(H) The quantity of experimentation needed to make or use the invention based on the content of the disclosure.
Scope of the claimed genus and nature of the invention.
Claim 1 claims an antibody that binds PSMA which comprises a VH comprising SEQ ID NO:30 and a VL comprising SEQ ID NO:31, wherein within the VH and VL-CDR regions the X1-X12 variables are selectable from a series of amino acids. Claims 2-14 and 17-23 are dependent on claim 1 and also allow for variation of the CDR and are rejected.
Claims 22-23 are for methods of treating, inhibiting, or diagnosing a tumor in vivo comprising: i) a PSMA antibody defined above; ii) a nucleic acid composition encoding the antibody above; or iii) a non-human host comprising a nucleic acid that encodes the antibody above
State of the Relevant Art; level of one of ordinary skill; and level of predictability of the art.
1) The murine antibody 5D3 was known to the prior art (US 2019/0330367 Barinka C et al.), wherein Barinka taught the 5D3 antibody can effectively bind PSMA protein and PSMA expressed on tumor cells (Fig. 11 and 13). A structure activity relationship that would permit alterations of the 5D3 antibody VH and VL CDR was not known wherein binding, yield, stability, and antibody secretion were optimized was not known to the prior art.
The prior art taught CDR grafting may result in reduced target binding even if the VH–VL interface residues are preserved (Chiu ML et al. (Antibodies 2019, 8(4), 55), page 16, 2.1.4 Back Mutations section, paragraph 1). Chiu taught any CDR grafting protocol must include a step to identify FR positions that are critical for maintaining the CDR conformation (page 16, 2.1.4 Back Mutations section, paragraph 1 ). Chiu taught if a non-human residue in a critical position cannot be preserved because there are no such human sequences, one usually applies the so-called back mutation, i.e., a mutation of a residue in the human FR to the amino acid that occurs in the non-human parent (page 16, 2.1.4 Back Mutations section, paragraph 1). Chiu taught back mutations reduce the humanness score of the resulting variant, but the change should improve the binding affinity (page 16, 2.1.4 Back Mutations section, paragraph 1) and may further improve expression of the antibody variants (page 17, paragraph 3). Chiu taught the central zone around CDR-H3 remains problematic for accurate modeling and the lack of information on the CDR involvement in antigen binding often leads to an excessive number of back mutations in the humanized antibodies (page 17, paragraph 2). Chiu taught to avoid such outcomes, each potentially critical position should be tested for back mutation and only those mutations that affect binding should be incorporated into the final antibody (page 17, paragraph 2). Thus, individual back mutations affect target binding, expression, and humanness of the antibody.
Regarding the location of the back mutations, Chiu taught back mutations in the VH-VL interface and that are in direct contact with CDRs, or “Vernier zone” (Chiu Fig 7-8). The Vernier zone constitutes 30 residues, wherein 4 are considered part of HCDR1 in the Chothia and IMGT definitions and the remaining are divided between the VH and VL (page 16, 2.1.4 Back Mutations section, paragraph 2). Chiu taught a careful analysis of the importance of each Vernier zone position in the context of given CDRs and antibody–antigen interactions is the cornerstone of the humanization process (page 16-17, bridging sentence). Thus, individual back mutations at specific positions within a humanized antibody affect target binding, expression, and humanness of the antibody and individual mutations are required for generation of an effective antibody.
At the time of the filing of the instant application, it was well established in the art that the formation of an intact antigen-binding site in an antibody usually required the association of the complete heavy and light chain variable regions of a given antibody, each of which consists of three “complementarity determining regions” (“CDRs”) which provide the majority of the contact residues for the binding of the antibody to its target epitope. E.g., Almagro & Fransson, Frontiers in Bioscience 2008; 13:1619-33; (see Section 3 “Antibody Structure and the Antigen Binding Site” and Figure 1). While affinity maturation techniques can result in differences in the CDRs of the antibody compared to its parental antibody (page 3 “The IgG Molecule, second and third paragraphs), those techniques involve trial-and-error testing and the changes that maintain or improve affinity are not predictable a priori. E.g., id., (page 6 ending paragraph onto page 7). Chiu ML et al. (Antibodies 2019 8, 55, 1-80) taught the antigen binding of antibodies often results in conformational changes in the contact surface areas of both the antibody and the antigen (page 5, first paragraph). Chiu further taught antibody modeling has been shown to be accurate for the framework region sequences, but CDR modeling requires further development and improvements (page 6, second paragraph).
In addition to changes within the CDR altering target binding, alterations to the CDR have been shown to dramatically alter antibody secretion. Hasegawa H et al. (mAbs 2017, 9(5) 854-873) taught a pair of human IgG clones with a single amino acid substitution in the variable region was sufficient to alter the efficiency of immunoglobulin biosynthesis (page 866, last sentence left column). Hasegawa taught the 2 mAbs differed only by one amino acid in the LC's CDR1 and that despite the near-identity of their primary sequences, the parental mAb secreted copious amounts of IgG to the culture media, while the variant mAb induced RB phenotypes extensively and secreted 20-fold less IgG (page 866, right column, first paragraph). Importantly, the 2 model IgGs were by no means abnormal or defective as mAbs, but demonstrated a profound impact of a single amino acid substitution on immunoglobulin biosynthesis (page 866, right column, first paragraph).
2) The murine antibody 5D3 was known to the prior art (US 2019/0330367 Barinka C et al.), wherein Barinka taught the 5D3 antibody can effectively bind PSMA protein and PSMA expressed on tumor cells (Fig. 11 and 13). Banerjee et al., (Journal of Nuclear Medicine 2019 60.3: 400-406 IDS reference) taught 111In-DOTA-5D3 is a radiolabeled antibody for imaging and a surrogate for therapy of malignant tissues expressing PSMA (abstract). Huang C et al. (Mol Pharm. 2020 Aug 17;17(9):3392–3402) taught anti-PSMA monoclonal antibodies (mAbs) have been developed as bioligands for diagnostic imaging and targeted PC therapy (abstract). Huang taught administration of an antibody drug conjugate of 5D3-DM1 to a subject with PSMA expressing cancer could effectively decrease tumor growth (abstract). The parental 5D3 antibody is not known to effectively treat subjects with cancer without a toxin conjugated to the antibody. The instant humanized 5D3-6 and variant antibodies in the instant specification would not be expected to effectively treat PSMA expressing tumors without being conjugated to a toxic agent.
Administration of antibodies and plasmids that encode antibodies is known to the prior art. Kim H et al. (Cancer Gene Therapy 2016 23, 341–347) taught administration of antibodies or plasmids encoding antibodies as an effective method to cause antibodies to enter the bloodstream in vivo (abstract). The prior art does not show a method of effectively administering a non-human host in vivo for diagnostic purposes.
The prior art has not described using a PSMA targeted antibody to diagnose a tumor that does not express PSMA.
Summary of Species disclosed in the original specification; the amount of direction provided by the inventor, existence of working examples; and quality of experimentation needed to make or use the invention based on the content of the disclosure.
1) The murine antibody 5D3 was known to the prior art (US 2019/0330367 Barinka C et al.), wherein Barinka taught the 5D3 antibody can effectively bind PSMA protein and PSMA expressed on tumor cells (Fig. 11 and 13). In the instant application the murine antibody 5D3 was humanized and back mutations were introduced into each model based on a visual inspection of the aligned structures of the 5D3 variable domains and search hits in order to avoid local structural incompatibilities between CDRs of the mouse donor antibody and the human acceptor framework regions (specification, page 30 last paragraph to page 31 first paragraph). Humanization of 5D3 produced three separate antibodies, namely 2-5D3, 5-5D3, and 6-5D3 wherein amino acid grafting of the CDRs of 5D3 and humanization produced antibodies with variable properties ( specification page 33, Table 2).
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Alignment of Kabat defined VH and VL CDRs with Clustal Omega showed that the CDRs were the same.
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While the CDR was maintained: 1) 2-5D3 and 5-5D3 had diminished yield (specification page 33, Table 2); 2-5D3 had stability issues (Fig. 9); and 3) 5-5D3 lacked specificity (Fig. 11). Only 6-5D3 was able to maintain acceptable yield, stability, and specificity.
The instant specification taught three humanized variants of a 5D3 murine antibody, wherein the 6-5D3-lgG1 (6-5D3-hFab) performs well as it is the only one that retained high expression yields, temperature stability and specificity of the parent 5D3 mAb. It was unexpected that a humanized variant of 5D3 murine antibody can be obtained which retains these three favorable characteristics of the parent 5D3 mAb despite the CDR-grafting during humanization (page 33, second paragraph).
Alignment of 5D3 and humanized 6-5D3 which includes back mutations show about an 73% identical match with the VH of 5D3 and an 88% match with the VL of 5D3. Mutations in 6-5D3 further fall within the Vernier Zone that include VH G49S, L69T, and A78L.
VH
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VL
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Further, in silico humanization of the 5D3 antibody using BioPhi an in silico antibody humanization tool available after the priority date was unable to identify the humanization sequence claimed (BioPhi et al. https://biophi.dichlab.org/humanization/humanize/ 2026).
VH 5D3-6 vs humanized in silico
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The instant specification taught generation of 6-5D3 variant clones LVg2, HVg2, HVg3, HVg4, HVg5, HVg7, LVg2HVg2, LVg2HVg3, LVg2HVg4, LVg2HVg5, and LVg2HVg7, wherein site-directed mutagenesis was performed at different locations throughout the VH and VL CDR. Table 4 identifies the locations of amino acid mutations tested as VH 30, 55, 56, 57, 62 and VL 31. In claim 1, the locations of the tested antibody mutants in a VH comprising SEQ ID NO:30 and a VL comprising SEQ ID NO: 31 are indicated as X1, X5-X7, and X10-X11. No mutations were tested at VH 31-32, 34, 58, 61, and VL 31 indicated as X2-X4, X8-X9, and X12 in a VH comprising SEQ ID NO:30 and a VL comprising SEQ ID NO: 31 in claim 1.
Further, only a small subset of amino acids were exchanged in comparison to the amino acids claimed in claim 1. See table below.
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The Applicant has enablement of 5D3-6 and its variants LVg2, HVg2, HVg3, HVg4, HVg5, HVg7, LVg2HVg2, LVg2HVg3, LVg2HVg4, LVg2HVg5, and LVg2HVg7 for individual antibody clones with the following X1-X12, wherein the amino acids from separate mutants are not mixed between clones.
5D3-6 N, T, S, M, D, G, D, T, N, G, N, N
LVg2 N, T, S, M, D, G, D, T, N, G,E, N
HVg2 Q, T, S, M, D, A, D, T, N, A, N, N
HVg3 Q, T, S, M, E, G, E, T, N, A, N, N
HVg4 Q, T, S, M, E, G, D, T, N, A, N, N
HVg5 Q, T, S, M, E, G, S, T, N, A, N, N
HVg7 Q, T, S, M, D, G, D, T, N, G, N, N
LVg2HVg2 Q, T, S, M, D, A, D, T, N, A, E, N
LVg2HVg3 Q, T, S, M, E, G, E, T, N, A, E, N
LVg2HVg4 Q, T, S, M, E, G, D, T, N, A, E, N
LVg2HVg5 Q, T, S, M, E, G, S, T, N, A, E, N
LVg2HVg7 Q, T, S, M, D, G, D, T, N, G, E, N
Alterations of CDR residues were shown in Table 4 to alter antibody binding. It is unknown if antibody yield and specificity were altered as shown in other humanized antibodies above.
Mixture of CDR residues that were tested could potentially have very large effects as seen when comparing HVg5 and LVg2HVg3, wherein changing 2 of the CDR residues show about a 50-fold change in antibody affinity
Clone X1-X10 ELISA (nM)
HVg5 Q, T, S, M, E, G, S, T, N, A, N, N 0.06
LVg2HVg3 Q, T, S, M, E, G, E, T, N, A, E, N 2.69
As noted below, the art generally accepted that the combination of the CDRs within the VH and VL pair of an antibody were essential for binding specificity. The specification does not describe what residues within the CDRs confer the binding activity claimed without altering antibody secretion, stability, or specificity and the claim language permits changes in the VH and VL that contain the CDRs in the VH or VL. Accordingly, the skilled artisan would not be able to discern a structure/function correlation for antibodies other than those comprising all six CDRs of a PSMA antibody of 5D3-6, and its variants LVg2, HVg2, HVg3, HVg4, HVg5, HVg7, LVg2HVg2, LVg2HVg3, LVg2HVg4, LVg2HVg5, and LVg2HVg7.
2) The instant specification taught 5D3-6, and its variants LVg2, HVg2, HVg3, HVg4, HVg5, HVg7, LVg2HVg2, LVg2HVg3, LVg2HVg4, LVg2HVg5, and LVg2HVg7y effectively bind PSMA in Fig. 10 and 16. The instant specification does not have examples of successfully: A) treating or inhibiting a tumor in vivo; or B) diagnosing a tumor in vivo: a) that is “used” rather than ---administered---; b) that does not express PSMA; or c) administering a non-human host.
Conclusion
Claims 1-14 and 17-23 are rejected.
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/J.J.S./Examiner, Art Unit 1643
/Karen A. Canella/Primary Examiner, Art Unit 1643
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