DETAILED ACTION
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 .
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
Priority
The instant application, filed 03/14/2023, is a 371 filing of PCT/US2021/041608, filed 09/27/2021, and claims domestic benefit to US provisional application 63/085,931, filed 09/30/2020.
Status of Claims/Application
The preliminary amendment of 10/18/2024 is acknowledged. Claims 1-539 are cancelled and claims 540-559 are currently pending and are examined on the merits herein.
Information Disclosure Statement
The information disclosure statements (IDS) submitted on 03/14/2023 and 12/03/2024 are in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statements have been considered by the examiner.
Drawings
The drawings filed 03/14/2023 are objected to because the figures are low resolution making them difficult to read. For instance, see Figs 1A-F, 3A-I, 4A-H, 5D-C, 7A-C, 8A-G, 10A-B, 11, 12A-B, 13A-C, 14A-B, 15A-D, 16A-D, 17A-B, 18, and 19.
Corrected drawing sheets in compliance with 37 CFR 1.121(d) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. The figure or figure number of an amended drawing should not be labeled as “amended.” If a drawing figure is to be canceled, the appropriate figure must be removed from the replacement sheet, and where necessary, the remaining figures must be renumbered and appropriate changes made to the brief description of the several views of the drawings for consistency. Additional replacement sheets may be necessary to show the renumbering of the remaining figures. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
Claim Objections
Claim 545 is objected to for the following informality: The claim is missing a period at the end of the claim.
Claim 557 is objected to for the following informality: The claim currently recites “the CAR polypeptide comprising: anti-MUC18 antibody comprises a variable heavy and light chain amino acid”. As the claim is not referencing a previous recitation of an anti-MUC18 antibody, the recitation of “anti-MUC18 antibody” should be preceded with “an” and the word “comprises” should be “comprising”. The claim should be modified to read “an anti-MUC18 antibody comprising” for grammatical correctness. 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 542-546, 549, and 557-559 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.
Claims 542, 544-546, and 557-559 recite sequences “having at least 80% sequence identity” to the recited SEQ ID NOs “or a fragment thereof”. Specifically, see the following:
Claim 542: lines 5-11; Claim 544, lines 1-4; Claim 545, lines 5-6; Claim 546, lines 1-3; Claim 557: lines 3-8; and claim 559, lines 2-7.
The recitations of both “having at least 80% sequence identity” and “or a fragment thereof” in the claims renders the metes and bounds of the claims indefinite as it is unclear how the limitations relate to each other. For instance, it is unclear if the fragments must have 80% identity to the recited sequence or if the claim encompasses 80% sequence identity to fragments of the recited sequences.
Claim 558 is rejected by virtue of its dependency on claim 557 as it does not resolve the ambiguity discussed above.
Appropriate correction is required.
Claim 543 recites the limitation "the one or more extracellular spacers of the first CAR" in line 7. There is insufficient antecedent basis for this limitation in the claim. Neither claim 543, nor claim 540 on which 543 depends, recites one or more extracellular spacers of the first CAR that could be being referenced rendering the metes and bounds of the claim indefinite.
Appropriate correction is required.
Claim 545 is missing a conjunction, such as “and” or “or” between the limitations regarding the extracellular domain (lines 2-6) and the CD3 zeta signaling domain (line 7) rendering the metes and bounds of the claim indefinite as the scope of the claim is unclear.
Additionally, the claim terminates with “or”. It is unclear if limitations were inadvertently left out or if the claim is meant to terminate following the limitation regarding the SEQ ID NO of the CD3 zeta signaling domain.
Additionally, the claim recites “the CD3 zeta signaling domain comprises SEQ ID NO: 16”. The claim depends on claim 540, which recites two CD3 zeta signaling domains, specifically one in the first CAR and one in the second CAR. As it is unclear which of the two CD3 zeta signaling domains are being limited to SEQ ID NO: 16, the metes and bounds of the claim is indefinite. See MPEP 2173.05(e) which states “ if two different levers are recited earlier in the claim, the recitation of "said lever" in the same or subsequent claim would be unclear where it is uncertain which of the two levers was intended. “
In the instant office action, claim 545 is interpreted as requiring the recited extracellular domain limitations “or” the CD3 zeta signaling domain recited. Additionally, the limitations regarding the CD3 zeta signaling domain is interpreted to mean that at least one of the CD3 zeta signaling domain in claim 540 is SEQ ID NO: 16.
Appropriate correction is required.
Claim 549 recites that the immune effector cells are NK cells in lines 1-2 and that the expression of the first and second CAR polypeptides by “an immune effector cells” increases the likelihood of NK activation at tumor cites in lines 2-3. Based on the use of “an” in lines 2-3, it is unclear if the immune effector cells are limited to NK cells as is previously recited in the claim or if the immune cell could be any type of immune cell.
Appropriate correction is required.
Claim Rejections - 35 USC § 112(a)
The following is a quotation of the first paragraph 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 the first paragraph of pre-AIA 35 U.S.C. 112:
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 542, 544, 546, and 557-559 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.
Claim 542 depends on claim 540 and recites that the one or more cancer associated antigens is MUC18 bound by an anti-MUC18 antibody. The claim further limits the variable heavy chain and variable light chain amino acid sequences as having “at least about 80% sequence identity” to SEQ ID NOs: 2 and 4, respectively “or a fragment thereof”.
Claim 544 depends on claim 540 and limits the first CAR polypeptide to comprising “an” amino acid sequence having “at least 80% sequence identity” to SEQ ID NOs: 12-15 “or a fragment thereof”.
Claim 557 recites a composition comprising a CAR comprising an anti-MUC18 antibody comprising a viable heavy chain and variable light chain amino acid sequences as having “at least about 80% sequence identity” to SEQ ID NOs: 2 and 4, respectively “or a fragment thereof”.
Claim 559 ultimately depends on claim 557, and further limits the CAR polypeptide to comprising “an” amino acid sequence having “at least 80% sequence identity” to SEQ ID NOs: 12-15 “or a fragment thereof”.
Claims 542 and 547 are drawn to a genus of heavy and light chain variable regions which have up to 20% modification (80% identity) in the claimed sequences as well as fragments of the claimed sequences which, in the broadest reasonable interpretation of the claim, encompasses small portions of the variable regions. The claims further limit this genus to performing the function of binding to MUC18 as an anti-MUC18 antibody.
Claims 544 and 559 are drawn to a genus of CAR polypeptides having up to 20% modification (80% identity) in the claimed sequences as well as fragments of the claimed sequences which, in the broadest reasonable interpretation of the claim, encompasses small portions of the variable regions. The claim also recites “an” amino acid sequence which, in the broadest reasonable interpretation, encompasses CARs comprising only one of the amino acids in the claimed sequences. The claims further limit this genus to performing the function of binding to one or more cancer-associated antigens or a binding region thereof.
The instant disclosure, however, does not describe a representative number of species of the claimed genus performing the claimed functions, nor does the disclosure identify a structure-function relationship that could be used to predictably identify which of the claimed amino acid sequences could modified or fragmented in what way while maintaining the claimed functions. This is particularly the case as the claims do not require a full complement of 6 CDRs, specifically 3 from the heavy chain variable region and 3 from the light chain variable region, which are the art recognized binding region of antibodies and antibody fragments.
The examples of the instant disclosure studied the effects of NK cells modified with a retroviral vector to express a chimeric NKG2D receptor (NKG2D.ζ), which comprises the extracellular domain of the native NKG2D molecule fused to the intracellular cytotoxic ζ-chain of the T cell receptor (page 126, [0495]). The examples further studied the use of the NKG2D.ζ receptor in combination with GD2.CAR-T cells. In the study, NKG2D.ζ NK cells were infused followed by GD2.CAR T cells (page 131, [0504]). Further studied is the combination of NKG2D.ζ with a MUC18-targeting CAR (page 136, Example 2, [0515]).
The examples of the instant disclosure demonstrate the application of a single species of the claimed antibody/CAR binding to a cancer associated antigen and/or MUC18. As such, the disclosure does not provide a representative number of species of the antibody/CAR where the heavy and light chain variable regions have up to 20% modification or comprise only a fragment of the claimed sequences while maintaining binding function.
The state of the art around the effective filing date of the claimed invention also does not provide a representative number of species or a predictable structure-function relationship to support the full scope of the claimed genus.
For example, Chiu, M.L., et al (2019) Antibody structure and function: The basis for engineering therapeutics Antibodies 8(55); 1-80 teaches that, the antigen-binding site of immunoglobulins is formed by the pairing of the variable domains (VH and VL) of the Fab region. Chiu teaches that each domain contributes three complementarity determining regions (CDRs), specifically, three from the VL and three from the VH, and that the six CDR loops are in proximity to each other resulting from the orientation of the VL and VH regions. Chiu teaches that the configuration of the VL and VH brings the three CDRs of the VL and VH domains together to form the antigen-binding site (page 4, paragraph 2). These teachings of Chiu demonstrate that the interaction between the heavy and light chain variable domains effect the conformation of the binding region of the antibody and therefore the antibody’s ability to bind to its target. Furthermore, the teachings of Chiu point out that the binding site is formed by the combination of the heavy and light chain CDRs (six regions) together. Based on these teachings, an ordinarily skilled artisan would not have been able to predictably identify which species of the instantly claimed genus would be capable of performing the claimed functions. This is particularly the case in the absence of a full complement of heavy and light chain CDRs.
Rabia, L., et al (2018) Understanding and overcoming trade-offs between antibody affinity, specificity, stability, and solubility Biochem Eng. J. 15(137); 365-374 discusses similar challenges faced during antibody optimization. Rabia discusses the challenges with optimizing antibody properties and states that “natural antibody affinity maturation relies on the introduction of somatic mutations followed by clonal selection of antibody variants with improved affinity. However, not all somatic mutations contribute to antibody affinity… antibodies accumulate some somatic mutations to increase affinity and others to compensate for the destabilizing effects of affinity-enhancing mutations” (page 2, paragraph 4). Rabia further provides an example of researchers who introduced mutations throughout variable frameworks and CDRs and created libraries to sort antibody variants with high antigen binding. In this case an antibody was identified that displayed increased affinity but had a significant reduction in stability (page 3, paragraph 2). Rabia concludes by stating that “a final key area of future work is the development of improved computational methods for predicting mutations in antibody CDRs and frameworks that co-optimize multiple antibody properties” and that “future efforts will also need to improve structural predictions of antibody CDRs – especially the long and highly variable heavy chain CDR3 – to accurately predict CDR mutations that are beneficial to different antibody properties” (page 9, paragraph 4 – page 10 paragraph 2).
Based on the teachings of Rabia, introducing mutations in the antibody structure, particularly in the CDR regions, is not a predictable task and requires experimentation following mutation to ensure that the binding affinity is maintained and a specific, stable antibody is created. Rabia further spoke to the use of libraries and computational methods for predicting and co-optimizing antibody properties and teaches that these methods are not robust enough yet to yield predictable results. These teachings demonstrate that a modification to even one amino acid of an antibody, particularly in the CDRs, would likely result in an antibody/CAR that is not suitable for binding to MUC18 or a cancer-associated antigen, as recited in the instant claims.
Rojas, G. (2022) Understanding and Modulating Antibody Fine Specificity: Lessons from Combinatorial Biology Antibodies 11(48); 1-22, which was published two years after the effective filing date of the claimed invention, demonstrates that antibody structure and function were still not predictable years after the effective filing date. For instance, Rojas teaches that epitope mapping results using mutagenesis scanning challenge our notions of conservative and nonconservative amino acid replacements. Several measures have been proposed to evaluate the difference between amino acids, based on physico-chemical distance between them, mutational distance, or evolutionary exchangeability. Tolerability profile to mutations within functional epitopes does not adjust strictly to any of these rules. The critical attributes of each amino acid that should be kept to maintain recognition depend on the particular antibody. For instance, sometimes only tyrosine and phenylalanine residues can be exchanged without effecting antigenicity, pointing to the relevance of their almost-identical aromatic rings, whereas in other epitopes, tyrosine and histidine are exchangeable, reflecting that two different rings can fulfill a similar functional role (page 11, paragraph 1). Teachings which demonstrate that even years after the effective filing date of the claimed invention even modifications using conservative substitution were not predictable.
It is not evident from the disclosure, or the prior art, that applicant was in possession of a representative number of species supporting the entire genus of antibodies/CDRs that are encompassed by the instant the claims. Additionally, there is no disclosed or art recognized structure-function relationship between antibody structure and functionality which would allow for the predictable modification or fragmentation of the claimed sequences while maintaining the claimed binding functions. Therefore, the instant claims were found to not meet the written description requirement.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 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 the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 540-541, 543-544, and 547-549 are rejected under 35 U.S.C. 103 as being unpatentable over US 2019/0255108 A1 (Yupo, M. et al) 22 Aug 2019.
US’108 teaches compositions and methods relating to chimeric antigen (CAR) polypeptides and engineered cells having CAR polypeptides directed to at least two targets (abstract). US’108 teaches compound CARs (cCAR) having at least two complete and distinct chimeric antigen receptor polypeptides. As used in the disclosure, “distinct chimeric antigen receptor polypeptides” has a unique antigen recognition domain, a signal peptide, a hinge region, a transmembrane domain, at least one costimulatory domain, and a signaling domain (page 19, [0291]).
US’108 teaches a CD123-NKG2D cCAR, a CLL-1- NKG2D cCAR, a CD33-NKG2D cCAR, and a BCMA-NKG2D cCAR (page 25, [0382]-[0388]).
US’108 teaches that NKG2D, the NKG2D receptor, is a transmembrane protein belonging to the CD94/NKG2 family of C-type lectin-like receptors. NKG2D can bind to at least 8 different ligands that are naturally expressed in AML, multiple myeloma, or other leukemias. NKG2D ligands are induced-self proteins which are virtually absent or present only at very low levels on the surface of normal cells, but are overexpressed in cancer cells. Therefore, they are good candidates for CAR targeting (page 25, [0382]).
A cCAR contains two units of CARs, for instance a CD123 CAR and NKG2D CAR that targets tumor cells expressing CD123 and NKG2D ligands, respectively (page 25, [0383]). US’108 teaches that the addition of NKG2D as a target to CD123, CLL-1, or CD33 CARs enhances the antitumor response and reduces the risk of antigen escape associated with disease relapse because NKG2D is widely expressed in AML, MDS, CML, and MPN. BCMA and NKG2D ligands are both widely expressed on multiple myeloma cells and this high expression allows the BCMA-NKG2D cCAR to have a comprehensive coverage of all potentially cancerous cells. This allows for a more complete elimination of cancerous cells to reduce antigen escape by hitting hard with multiple targets simultaneously before resistance develops (page 25, [0384]-[0388]).
While US’108 does not explicitly disclose that the cCARs CD123-NKG2D, CLL-1- NKG2D, CD33-NKG2D, and BCMA-NKG2D comprise the domains claimed in instant claim 540, the teachings of US’108 as a whole demonstrate that the claimed domains would have been obvious to use.
For instance, US’108 teaches that the CAR polypeptides have a signaling peptide, an antigen recognition domain, a hinge region, a transmembrane domain, at least one co-stimulatory domain, and a signaling domain (page 14, [0215]).
US’108 teaches that the target specific antigen recognition domain includes an antigen binding domain derived from an antibody against an antigen of the target or a domain derived from a receptor binding to a peptide or protein ligand on the target (page 14, [0221]). The antigen recognition domain can also include an antigen binding fragment including an scFv, which is a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of immunoglobulins connected with a short peptide linker (page 14, [0223]).
US’108 further teaches that the CARs can comprise a hinge region, including those selected from an immunoglobulin including IgG1 and IgG4 (page 15, [0228]-[0230]).
US’108 teaches that the intracellular signaling domain includes the polypeptide of a functional signaling domain, such as that of CD3 zeta (page 15, [0238]).
US’108 teaches that the costimulatory domain is a functional signaling domain selected from a protein including Cd28, 4-1BB, ICOS, OX40, CD30, CD40, 2B4, CD2, or LIGHT (page 15, [0238]).
US’108 further teaches polynucleotide sequences encoding the CARs and teaches that the polynucleotide can be easily prepared from an amino acid sequence of the specified CAR by any conventional method (page 15, [0239]). US’108 teaches that the polynucleotide can be cloned into a vector, which is a composition of matter including an isolated polynucleotide and can be used to deliver the isolated polynucleotide into the interior of a cell (page 16, [0242]). The vectors include expression vectors (page 16, [0243], [0247], and [0249]).
US’108 further teaches engineered cells having multiple CAR units allowing a single engineered cell to target multiple antigens. Targeting multiple surface markers or antigen simultaneously with a multiple CAR unit prevents selection of resistant clones and reduces tumor occurrence (page 19, [0297]). US’108 further teaches that engineered cells are cells that are modified, transformed, or manipulated by the addition or modification of a gene, a DNA or RNA sequence, or protein or polypeptide. Isolated cells, host cells, and genetically engineered cells include isolated immune cells, such as NK cells and T cells that contain the DNA or RNA sequences encoding the CARs on the cell surface. Isolated host cells and engineered cells may be used, for example, for enhancing the NK cell activity or a T lymphocyte activity, treatment of cancer, and treatment of infectious diseases (page 18, [0261]).
In a preferred embodiment, the engineered cell having at least two distinct chimeric antigen receptor polypeptides is a primary NK cell isolated from the peripheral blood or cord blood and NK-92 cells, such that it is administered “off-the-shelf” to an mammal with a disease or cancer (page 20, [0302]). US’108 further teaches that NK cells do not need pre-activation and constitutively exhibit cytolytic functions. Further expression of cCARs in NK cells allows NK cells to effectively kill cancers. Further, NK cells are known to mediate anti-cancer effects without the risk of inducing GVHD (page 37, [0614]-[0615]).
US’108 teaches that it was found that the compound CAR in T cells or NK cells targeting different or the same tumor populations combat tumor factors that cause cancer cell resistant to CAR killing activity, such as downregulation of the target antigen from the cancer cell surface. It was also found that this downregulation enables the cancer cells to “hide” from the CAR therapy, referred to as “antigen escape”. The cCARs have significant advantages over single-CAR therapies due to its multi-targeting ability. While loss of a single antigen under antigen-specific selection pressure is possible, loss of two major antigens simultaneously is much less likely (page 20, [0313]). US’108 further teaches that compound CARs exhibit less toxicity compare to a single CAR. A compound CAR increases the affinity or trafficking to the tumor cell expressing two target antigens rather than off-target cells that only express one target antigen. In this way, the compound CAR may elicit selectivity and prefer to target cells expressing both target antigens rather than cells expressing only one antigen (pages 24-25, [0375]).
It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention to modify the CD123-NKG2D, CLL-1- NKG2D, CD33-NKG2D, and BCMA-NKG2D cCARs of US’108 to comprise the domains of the instant claims because US’108 teaches such domains for use in the cCARs disclosed. Thus, and ordinarily skilled artisan would have had a reasonable expectation of success.
Regarding claim 541, in addition to CD123, CLL-1, CD33, and BCMA, US’108 teaches that the first antigen recognition domain can alternatively target antigens including GD2, PSCA, WT1, CEA, HER-2, MAGE-3, MAGE-4, MAGE-5, MAGE-6, NY-ESO, c-Met, MUC1, EGFRvIII, and NKG2D (page 20, [0304]).
It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention to modify the CD123-NKG2D, CLL-1- NKG2D, CD33-NKG2D, and BCMA-NKG2D cCARs of US’108 by substituting CD123, CLL-1, CD33, and BCMA with an antibody or antibody fragment that binds to one of the alternative antigens disclosed by US’108. An ordinarily skilled artisan would have been able to substitute the antigen binding domains with a reasonable expectation of success as US’108 teaches that the antigens are alternative targets to CD123, CLL-1, CD33, and BCMA.
Regarding claim 544, in the CAR constructs taught by US’108, the linker peptide used to connect the VH and VL regions into an scFv is GGGGSGGGGSGGGGS and the CD8 leader has the sequence MALPVTALLLPLALLLHAARP, for instance see the CARs of SEQ ID NOs: 3, 18, 30, 36, and 42, both of which are identical to those used in the CARs of SEQ ID NOs: 12-15. The sequences of the CD8 leader and linker are fragments of instant SEQ ID NOs: 12-15 and, therefore, meet the instant claim limitations of the first CAR comprising SEQ ID NOs: 12-15, or a fragment thereof, respectively. It is noted that the disclosure defines “polypeptide fragment” or “fragment” on page 59, [0080] and states that fragments are typically “at least 5, 6, 8, or 10 amino acids long” indicating that each of the above sequences meet the limitation of a fragment of SEQ ID NOs: 12-15.
Claims 540-541, 543-545, 547-550, and 552-556 are rejected under 35 U.S.C. 103 as being unpatentable over WO 2020/132190 A1 (Hou, B., et al) 25 JUN 2020 in view of US 2019/0255108 A1 (Yupo, M. et al) 22 Aug 2019 and US 10,336,804 B2 (Zhang, T. and C.L. Sentman) 2 Jul 2019.
WO’190 teaches antibodies that bind to MUC18, also known as CD146 or melanoma cell adhesion molecule (MCAM). MUC18 is a transmembrane glycoprotein that functions primarily in cell adhesion (page 1, lines 4-5).
WO’190 further teaches chimeric antigen receptors (CAR) targeting MUC18 and immune cells expressing the CARs. The CARs are artificial cell-surface receptors that redirect binding specificity of immune cells to MUC18+ expressing cells, such as epithelium-derived cancer cells, thereby eliminating the target disease cells via, e.g., the effector activity of the immune cell. A CAR construct often comprises an extracellular antigen binding domain fused to at least an intracellular signaling domain. The extracellular antigen binding domain, which can be a single-chain antibody fragment (scFv), is specific to a MUC18 antigen and the intracellular signaling domain can mediate a cell signal that leads to activation of the immune cells. As such, immune cells expressing a car construct specific to MUC18 can bind to diseased cells, such as tumor cells, expressing MUC18, leading to activation of the immune cells and elimination of the diseased cells (page 27, lines 7-18).
WO’190 teaches that the disclosed anti-MUC18 antibodies can be used to produce the CAR constructs. For example, the VH and VL domains of the antibody can be fused to the intracellular signaling domain(s) to produce a CAR construct using conventional recombinant technology. In some examples, the VH and VL domains are connected via a peptide linker to form an scFv fragment (page 27, lines 19-23).
The CAR constructs may comprise one or more intracellular signaling domains. In some examples, the CAR comprises an intracellular signaling domain that includes an immunoreceptor tyrosine-based activation motif (ITAM). Such an intracellular signaling domain may be from Cd3ζ. In addition, the CAR may further comprise one or more co-stimulatory domains, which may be from a co-stimulatory receptor, for example, from 4-1BB (CD137), CD7, CD27, CD28, CD40, OX40, ICOS, GITR, HVEM, TIM1, or LFA-1 (page 27, lines 24-30).
The CAR may further comprise a transmembrane-hinge domain, which can be obtained from a suitable cell surface receptor, for example CD28 or CD8 (page 28, lines 1-3).
Also provided are isolated nucleic acid molecules and vectors encoding any of the anti-MUC18 CARs and host cells, such as host immune cells, e.g., T cells and natural killer cells, comprising the nucleic acid molecules or vectors. Immune cells expressing anti-MUC18 CARs, can be used for the treatment of cancers that express MUC18. Thus, also provided are methods of treating a subject with MUC18+ cancer, by selecting a subject with a cancer that expresses MUC18 and administering to the subject a therapeutically effective amount of the immune cells expressing the MUC18-targeted CAR (page 28, lines 4-11).
WO’190 further teaches pharmaceutical compositions comprising the immune cells expressing MUC18-targeting CARs mixed with a pharmaceutically acceptable carrier/excipient to form compositions for use in treating a target disease (page 28, lines 13-18).
WO’190 teaches that the immune cells, e.g., T cells and NK cells, expressing the anti-MUC18 targeting CARs are useful for inhibiting and/or eliminating MUC18+ disease cells, such as MUC18+ cancer cells, thereby benefiting treatment of a disease or disorder associated with the MUC18+ disease cells (page 32, lines 1-8). To practice the method, an effective amount of the pharmaceutical composition is administered to a subject in need of treatment via a suitable route (page 32, lines 9-14). A human subject who needs the treatment may be a human patient having, at risk for, or suspected of having a target disease/disorder associated with MUC18+ disease cells. In some embodiments, the MUC18+ disease cells are cancer cells, for example, epithelial cancer cells, i.e., derived from epithelial cells. Examples include, but are not limited to, ovarian cancer cells, breast cancer cells, renal cancer cells, lung cancer cells, colorectal cancer cells, and brain cancer cells (page 32, lines 19-27). WO’190 further teaches the treatment of melanoma (page 46, Example 3; page 1, lines 1-10). It is noted that the instant specification states “wherein the carcinoma comprises melanoma” (page 10, [0027]), indicating that melanoma meets the limitations of the cancer being a carcinoma. Additionally, carcinomas are cancer cells derived from epithelial cells indicating that the cancers taught by WO’190 also meet the limitation of the cancer being a carcinoma.
WO’190 further teaches that, when immune cells expressing a MUC18-targeting CAR are used for disease treatment, patients can be treated by infusing therapeutically effective doses of such immune cells, such as T cells or NK cells, in the range of about 105 to 1010 or more cells per kilogram of body weight (cells/Kg). This infusion can be repeated as often and as many times as the patient can tolerate until a desired response is achieved. The appropriate infusion dose and schedule will vary from patient to patient, but can be determined by the treating physician for a particular patient. Typically, initial doses of approximately 106 cells/Kg will be infused, escalating to 108 or more cells/Kg (page 38, lines 17-26). The dosages taught by WO’190 overlap with (105 to 1010 cells/kg of body weight) or lie inside of (106 cells/kg and 108 cells/Kg) the range claimed in instant claim 553 rendering the claimed range obvious per MPEP 2144.05 (I).
WO’190 exemplifies preparation and in vitro evaluation of anti-MUC18 CAR+ T cells in Example 2, starting on page 45. In the example, humanized CL070325 anti-MUC18 antibodies (VH and VL) were inserted into a chimeric antigen receptor vector in frame with a CD8 hinge region, a CD8 transmembrane region, an intracellular domain of the co-stimulatory 4-1BB, and an intracellular domain of CD3ζ. The vector further comprises an EF1α promotor. Nucleic acids encoding the anti-MUC18 CAR+ were transfected using lentiviral packaging into 293T cells. Human CD3+ T cells were then activated and transduced with lentivirus to produce anti-MUC18 CAR+ T cells (page 45, lines 16-23). The ability of the CAR T cells to induce antigen dependent cytotoxicity and IFNγ secretion in MUC18+ melanoma cell lines was studied. Specific lysis/cytotoxicity was also assessed. In all MUC18+ cell lines, the addition of the CAR T cells induced antigen dependent cytotoxicity compared to control experiments that led to no significant induction of specific toxicity (page 45, line 16 – page 46, line 18). WO’190 further performed in vivo evaluation of the anti-MUC18+ CAR T cells (page 46, starting on line 20). Mice treated with the anti-MUC18 CAR+ T cells had a reduction in tumor growth relative to controls. Sustained reduction in melanoma cells were observed through day 21 of the experiment (page 46, line 20 – page 47, line 10).
WO’190 differs from the instantly claimed invention in that WO’190 does not disclose that the anti-MUC18 CAR is in a composition with a second CAR polypeptide comprising an NKG2D receptor or fragment thereof and a CD3 zeta signaling domain.
The teachings of US’108 are as discussed in detail above.
US’804 teaches a chimeric receptor molecule composed of a natural killer cell receptor and an immune signaling receptor expressed on the surface of a T cell to activate killing of a tumor cell. Nucleic acid sequences encoding the chimeric receptor molecule are introduced into T cells ex vivo and T cells that express the chimeric receptor molecule are subsequently injected into a subject in need of treatment. In this manner, the chimeric receptor molecules provide a means for the subject’s own immune cells to recognize and activate anti-tumor immunity and establish long-term, specific, anti-tumor responses for treating tumors or preventing regrowth of dormant or residual tumor cells (col. 4, lines 13-32).
By way of illustration, murine chimeric receptor molecules composed of NKG2D or Dap 10 in combination with a N-terminally attached CD3ζ were generated and expressed in murine T-cells. NKG2D is a type II protein, in which the N-terminus is located intracellularly, whereas the CD3ζ chain is a type I protein with the C-terminus in the cytoplasm. To generate a chimeric NKG2D- CD3ζ fusion protein, an initiation codon, ATG, was placed ahead of the coding sequence for the cytoplasmic region of the CD3ζ chain (without a stop codon TAA) followed by a wild-type NKG2D gene. Upon expression, the orientation of the CD3ζ portion is reversed inside of the cells. The extracellular and transmembrane domains are derived from NKG2D (col. 4, lines 33-51).
US’804 further teaches that, in humans, two families of ligands for NKG2D have been described. NKG2D binds to the polymorphic MHC class I chain-related molecules (MIC)-A and MICB. These are expressed on many human tumor cell lines, on several freshly isolated tumor specimens, and at low concentrations on gut epithelium. NKG2D also binds to another family of ligands designated as the UL binding proteins (ULBP)-1, -2, and -3 molecules (col. 3, lines 26-36). US’804 further demonstrates lysis of cells transduced with MIC-A with the NKG2D-transduced immune cells (col. 8, lines 43-55) further demonstrating binding of the ligands.
US’804 teaches that, to test whether murine chimeric NKG2D-transduced T cells were capable of recognizing NKG2D ligands, NKG2D ligand-positive tumor cells were used as targets for chimeric NKG2D bearing T cells. The chimeric NKG2D transduced T cells produces high amounts of IFNγ after co-culture with RMA/Rae-1β, RMA/H60, or YAC-1 cells, compared to RMA cells with no ligands, indicating that the chimeric NKG2D modified T cells could functionally recognize NKG2D bearing tumor cells. Similarly, chimeric human NKG2D-bearing CD8+ T cells secrete IFNγ when brought into contact with human tumor cells from breast cancer, prostate cancer, pancreatic cancer, and melanoma cancer (col. 5, line 32 – col. 6, line 20). In vitro cytotoxicity was also evaluated where NKG2D transduced T cells were able to lyse NKG2D ligand expressing target cells. US’804 teaches that the killing of tumor cells demonstrates that chimeric NK receptors provide the T cells with a means to kill tumor cells that express endogenous NKG2D ligands (col. 5, lines 20-52).
US’804 further teaches that, although specific immunity may be obtained against tumors, local immunosuppressive mechanisms, such as regulatory T cells, prevents the function of tumor-specific T cells. The ability to remove these cells and enhance local anti-tumor immune function would be of great benefit for the treatment of cancer. In addition to activating host immunity, it has also now been found that adoptive transfer of chimeric NK cell receptor bearing T cells can also eliminate host regulatory T cells. It was observed that treatment of mice with advanced tumors with chimeric receptor bearing T cells results in a rapid loss of FoxP3+ T regulatory cells from within the tumor microenvironment and a destruction of advanced tumor cells (col. 10, lines 4-19).
It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention to modify the MUC18 targeting CAR immune cells of WO’190 to further include a second CAR forming a compound CAR expressing immune cell as taught by US’108 and to have used the NKG2D chimeric receptor disclosed by US’804 as the second CAR which is further supported by US’108. An ordinarily skilled artisan would have been motivated to form a cCAR expressing immune cell in order to target multiple antigens simultaneously with a multiple CAR units preventing selection of resistant clones and reducing tumor occurrence as taught by US’108. US’108 also teaches that using cCARs can overcome antigen escape while also increasing specificity, affinity, and tracking to the tumor cells expressing both antigens, thereby reducing off-target toxicity compared to cells expressing only one CAR. An ordinarily skilled artisan would have further been motivated to use the NKG2D chimeric receptor as the second CAR in the cCAR as US’804 teaches that, when expressed in immune cells, the NKG2D chimeric receptors activate host immunity while also reducing immunosuppressive cells, such as Treg cells in the tumor microenvironment leading to destruction of advanced tumor cells. An ordinarily skilled artisan would have had a reasonable expectation of success as US’108 demonstrates the production of compound CARs and their expression in immune cells, including cCARs that target a tumor antigen as well as NKG2D. Additionally, WO’190 and US’804 teach that the MUC18 CAR and the NKG2D chimeric receptor are active in overlapping cancers including melanoma and breast cancer.
Regarding claim 544, in the CAR constructs taught by US’108, the linker peptide used to connect the VH and VL regions into an scFv is GGGGSGGGGSGGGGS and the CD8 leader has the sequence MALPVTALLLPLALLLHAARP, for instance see the CARs of SEQ ID NOs: 3, 18, 30, 36, and 42, both of which are identical to those used in the CARs of SEQ ID NOs: 12-15.
It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention to use the linker and CD8 leader sequences disclosed by US’108 in the construction of the CAR disclosed by the combination of WO’190, US’108, and US’804. An ordinarily skilled artisan would have been motivated to use these sequences as US’108 demonstrates them as functional sequences in the generation of cCAR domains. Thus one of ordinary skill in the art would have had a reasonable expectation of success. The sequences of the CD8 leader and linker are fragments of instant SEQ ID NOs: 12-15 and, therefore, meet the instant claim limitations of the first CAR comprising SEQ ID NOs: 12-15, or a fragment thereof, respectively. It is noted that the disclosure defines “polypeptide fragment” or “fragment” on page 59, [0080] and states that fragments are typically “at least 5, 6, 8, or 10 amino acids long” indicating that each of the above sequences meet the limitation of a fragment of SEQ ID NOs: 12-15.
Claim 542 is rejected under 35 U.S.C. 103 as being unpatentable over WO 2020/132190 A1 (Hou, B., et al) 25 JUN 2020 in view of US 2019/0255108 A1 (Yupo, M. et al) 22 Aug 2019 and US 10,336,804 B2 (Zhang, T. and C.L. Sentman) 2 Jul 2019, as applied to claims 540 and 541 above, and in further view of US 6,924,360 B2 (Green, L.L, an dM. Bar-Eli) 2 AUG 2005.
The combination of WO’190, US’108, and US’804 teach the composition of claim 541 as discussed in detail above.
As discussed above, WO’190 teaches that anti-MUC18 antibodies can be used to produce the CAR constructs. For example, the VH and VL domains are connected via a peptide linker to form an scFv fragment (page 27, lines 19-23).
US’108 also teaches that the antigen recognition domain of the CAR can include a single chain variable fragment (scFv) which is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a short linker peptide (page 14, [0223]).
In the CAR constructs taught by US’108, the linker peptide used to connect the VH and VL regions comprises GGGGSGGGGSGGGGS. For instance, see the CARs of SEQ ID NOs: 3, 18, 30, 36, and 42 all of which use the (G4S)3 linker to combine VH and VL regions to form scFvs. The linker used in US’108 to form scFvs is identical to instant SEQ ID NO: 3 as shown in the alignment below:
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The combination of applied references, however, does not disclose that the anti-MUC18 antibody comprises the heavy and light chain variable regions claimed.
US’360 teaches anti-MUC18 monoclonal antibodies and their use in the treatment of disorders associated with increased activity of MUC18 (abstract). US’360 teaches that MUC18 is expressed on a variety of malignant neoplasms including smooth muscle neoplasms, such as Leiomyomas and leiomyosarcomas; tumors of vascular origin, such as angiosarcomas and kaposi’s sarcomas; placental site trophoblastic tumors, choriocarcinomas, and melanomas (col. 1, lines 47-56).
US’360 teaches the anti-MUC18 antibody c3.19.1, also referred to as ABX-MA1 (col. 3, lines 40-42) which is disclosed as having a heavy and light chain variable region of SEQ ID NOs: 1 and 2, respectively (col. 4, lines 55-59).
The variable regions of c.3.19.1 are identical to those of instant SEQ ID NOs: 2 and 4 as shown in the alignments below:
US’360, SEQ ID NO: 1 aligned with instant SEQ ID NO: 2
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US’360, SEQ ID NO: 2 aligned with instant SEQ ID NO: 4
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It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention to substitute the VH and VL antibody domains in the MUC18 CAR taught by the combination of WO’190, US’108, and US’804 with the VH and VL anti-MUC18 antibody domains taught by US’360 and to form a scFv with the VH and VL domains using the linker taught by US’108. It would have been obvious to substitute the VH/VL antibody domains with those taught by US’360 as the antibody disclosed by US’360 also binds MUC18, which is the same binding target as the MUC18 CAR. An ordinarily skilled artisan would have had a reasonable expectation of success as the use of either antibody VH/VL regions would target and bind to MUC18. It would have further been obvious to use the peptide linker used by US’108 to form a scFv by linking the VH and VL domains as US’108 demonstrates the use of the peptide linker to fuse VH and VL domains for the formation of scFvs for use in CARs, which is the same general method taught by WO’190 for construction of MUC18 CARs.
Claim 544 is rejected under 35 U.S.C. 103 as being unpatentable over WO 2020/132190 A1 (Hou, B., et al) 25 JUN 2020 in view of US 2019/0255108 A1 (Yupo, M. et al) 22 Aug 2019 and US 10,336,804 B2 (Zhang, T. and C.L. Sentman) 2 Jul 2019, as applied to claims 540 above, and in further view of US 6,924,360 B2 (Green, L.L, an dM. Bar-Eli) 2 AUG 2005 and WO 2018/161017 (Suri, V. et al) 07 Sept 2018.
The combination of WO’190, US’108, and US’804 teach the composition of claim 540 as discussed in detail above.
As discussed in detail above, WO’190 exemplifies a MUC18 targeting CAR comprising anti-MUC18 antibody variable regions (VH and VL) inserted into a chimeric antigen receptor vector in frame with a CD8 hinge region, a CD8 transmembrane region, an intracellular domain of the co-stimulatory 4-1BB, and an intracellular domain of CD3ζ (page 45, lines 16-23). WO’190 also teaches that anti-MUC18 antibodies can be used to produce the CAR constructs. For example, the VH and VL domains are connected via a peptide linker to form an scFv fragment (page 27, lines 19-23). WO’190 further teaches that the transmembrane can be obtained from a suitable cell-surface receptor, for example CD28 or CD8 (page 28, lines 1-3).
US’108 further teaches that the antigen recognition domain of the CAR can include a single chain variable fragment (scFv) which is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a short linker peptide (page 14, [0223]). US’108 teaches that the level of the surface CAR expression on the T cells or NK cells is affected by an appropriate leader sequence (page 1, [0010]) and teaches CAR constructs comprising a CD8 leader sequence (page 2, [0027]).
In the CAR constructs taught by US’108, the linker peptide used to connect the VH and VL regions into an scFv is GGGGSGGGGSGGGGS and the CD8 leader has the sequence MALPVTALLLPLALLLHAARP. For instance, see the CARs of SEQ ID NOs: 3, 18, 30, 36, and 42.
The CD8 leader sequence disclosed in the CAR constructs of US’108 is identical to instant SEQ ID NO: 13 AA 1-21 as shown in the alignment below:
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As discussed