Immunotherapy targeting cd70

Abstract

Disclosed herein a polypeptides for use in chimeric antigen receptors (CARs) for binding to CD70-expressing cells. The polypeptides are variants of a natural ligand for CD70. CD70-targeting CARs demonstrate enhanced cytotoxic against CD70-positive cancer cells. The polypeptides may also be used to detect the presence of CD70-expressing cells that are implicated in disease.

Description

PATENT Attorney Docket No.081906-1470154-254010PC Client Ref. No. SF2023-176 IMMUNOTHERAPY TARGETING CD70 CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 595,231 filed November 1, 2023, the content of which is incorporated herein by reference in its entirety. BACKGROUND

[0002] CD70 is a highly abundant protein found to be enriched on the surface of certain cancer cells, for example cells implicated in leukemia, lymphoma, and multiple myeloma (MM). While current chimeric antigen receptor T-cell (CAR-T) and bispecific T-cell engaging (BiTE) antibody therapies have improved outcomes for patients of certain cancers, most patients will ultimately relapse. For example, CD19 or BCMA are cell-surface proteins that are highly expressed on B- cell neoplasms. While FDA-approved CAR-T therapies against CD19 and BCMA have markedly improved outcomes for patients, most patients will experience disease progression after receiving treatment (Cappell KM, Kochenderfer JN. Long-term outcomes following CAR T cell therapy: what we know so far. Nat Rev Clin Oncol.2023 Jun;20(6):359-371. In another example, nearly all patients of acute lymphoblastic leukemia / lymphoma (ALL / LBL) treated with CAR-T cell therapy have at least some less severe toxicity manifestations, while 23-46% of patients display severe supraphysiologic cytokine production and massive in vivo T cell expansion. Frey et al., Hematology Am Soc Hematol Educ Program 2016; 2016 (1): 567–572; doi.org / 10.1182 / asheducation-2016.1.567. Meanwhile, there are no approved CAR-T therapies for acute myeloid leukemia (AML), in part due to difficulty in identifying AML-specific targets which do not cause unacceptable toxicities to normal tissue when targeted with immunotherapies. SUMMARY

[0003] Provided herein are materials and methods related to targeting CD70-expressing cells with CD27 fragments and mutants thereof.PATENT Attorney Docket No.081906-1470154-254010PC Client Ref. No. SF2023-176

[0004] In one aspect, a nucleic acid is provided encoding a polypeptide that comprises an extracellular target-binding domain that binds to human CD70. In some embodiments the polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2. In some embodiments, the polypeptide does not comprise wildtype full-length CD27 (SEQ ID NO: 1). In some embodiments, the amino acid sequence is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1 and comprises at least one amino acid substitution compared to wildtype CD27.

[0005] In some embodiments, the amino acid sequence comprises the sequence of SEQ ID NO: 2 and an amino acid substitution at or corresponding to one or more of positions 63, 66, 89, 93, 94 and 97 of SEQ ID NO: 2. In some embodiments, the amino acid sequence comprises an amino acid substitution at position 66 selected from N66A, N66C, N66D, N66E, N66F, N66G, N66H, N66I, N66K, N66L, N66M, N66P, N66Q, N66R, N66S, N66T, N66V, N66W, and N66Y. In some embodiments, the amino acid sequence comprises an amino acid substitution at or corresponding to position 63 selected from R63A, R63C, R63D, R63E, R63F, R63G, R63H, R63I, R63K, R63L, R63M, R63N, R63P, R63Q, R63S, R63T, R63V, R63W, and R63Y. In some embodiments, the amino acid sequence comprises an amino acid substitution at or corresponding to position 89 selected from Q89A, Q89C, Q89D, Q89E, Q89F, Q89G, Q89H, Q89I, Q89K, Q89L, Q89M, Q89N, Q89P, Q89R, Q89S, Q89T, Q89V, Q89W, and Q89Y. In some embodiments, the amino acid sequence comprises an amino acid substitution at or corresponding to position 93 selected from K93A, K93C, K93D, K93E, K93F, K93G, K93H, K93I, K93L, K93M, K93N, K93P, K93Q, K93R, K93S, K93T, K93V, K93W, and K93Y. In some embodiments, the amino acid sequence comprises an amino acid substitution at or corresponding to position 94 selected from E94A, E94C, E94D, E94F, E94G, E94H, E94I, E94K, E94L, E94M, E94N, E94P, E94Q, E94R, E94S, E94T, E94V, E94W, and E94Y. In some embodiments, the amino acid sequence comprises an amino acid substitution at or corresponding to position 97 selected from E97A, E97C, E97D, E97F, E97G, E97H, E97I, E97K, E97L, E97M, E97N, E97P, E97Q, E97R, E97S, E97T, E97V, E97W, and E97Y.PATENT Attorney Docket No.081906-1470154-254010PC Client Ref. No. SF2023-176

[0006] In some embodiments, the polypeptide is a chimeric antigen receptor (CAR). In some embodiments, the CAR comprises a hinge domain, a transmembrane domain, and a signaling domain.

[0007] In some embodiments, the hinge domain comprises a human immunoglobulin hinge region, a hinge region from CD8 alpha, CD4, CD28, PD1, CD152, and CD7, or a mutant thereof. In some embodiments, the hinge domain is IgG4 EQ.

[0008] In some embodiments, the transmembrane domain comprises all or part of a transmembrane domain of the alpha, beta or zeta chain of the T-cell receptor, CD28, CD27, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD 11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2R beta, IL2R gamma, IL7R a, ITGAl, VLAl, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDl la, LFA-1, ITGAM, CDl lb, ITGAX, CDl lc, ITGB 1, CD29, ITGB2, CD 18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100, (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME, (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKG2D, or NKG2C.

[0009] In some embodiments, the hinge and transmembrane domains comprise a combined amino acid sequence of SEQ ID NO: 14 or 20.

[0010] In some embodiments, the signaling domain comprises a CD28 co-stimulatory domain and / or a CD3 zeta activation domain. In some embodiments, the signaling domain comprises an activation domain from CD3 zeta, common FcR gamma, Fc gamma Rlla, FcR beta (Fc Epsilon Rib), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, or DAP12. In some embodiments, the signaling domain comprises a co-stimulatory domain from CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, or a ligand that binds to CD83. In some embodiments, the co-stimulatory domain comprises an amino acid sequence of SEQ ID NO: 15 or 16.PATENT Attorney Docket No.081906-1470154-254010PC Client Ref. No. SF2023-176

[0011] In some embodiments, the CAR comprises an amino acid sequence that is at least 95% identical to the amino acid of SEQ ID NO: 12, 17, 22, 23, or 24. In some embodiments, the CAR comprises an amino sequence of SEQ ID NO: 12, 17, 22, 23, or 24.

[0012] Also provided is a polypeptide that comprises an extracellular target-binding domain that binds to human CD70 as described above or elsewhere herein.

[0013] In another aspect, a vector comprises a nucleic acid disclosed herein.

[0014] In another aspect, a cell comprises a polypeptide encoded by a nucleic acid disclosed herein. In some embodiments, the cell is a human T cell, natural killer (NK) cell, macrophage, monocyte, B cell, gamma / delta T cell, natural killer T (NKT) cells induced pluripotent stem cell, hematopoietic stem cell, myeloid progenitor cell, or lymphoid progenitor cell.

[0015] In another aspect, a composition comprises (a) a cell of the present disclosure and (b) a pharmaceutically acceptable carrier.

[0016] In another aspect, the present disclosure provides a method of introducing a nucleic acid encoding a polypeptide into a cell. In some embodiments, the method comprises: (a) providing the cell; and (b) introducing the nucleic acid or the vector of the present disclosure into the cell to form a cell expressing the polypeptide. In some embodiments, the cell is a human cell, optionally an immune cell, and the providing comprises obtaining the cell from a human. In some embodiments, the method further comprises introducing the cell expressing the polypeptide into a human. In some embodiments, the cell is introduced into the human from which the cell was obtained.

[0017] In another aspect, the present disclosure provides a method of treating cancer or an autoimmune disease in a patient, the method comprising administering to the subject a pharmaceutically effective amount of the composition of the present disclosure. In some embodiments, the cells of the cancer are CD70-expressing cells. In some embodiments, the cancer is leukemia, acute myeloid leukemia (AML), B cell acute lymphoblastic leukemia (B-ALL), B cell chronic lymphocytic leukemia (B-CLL), hairy cell leukemia, B-cell prolymphocytic leukemia, non-Hodgkin’s B cell lymphoma, diffuse large B cell lymphoma (DLBCL), follicular lymphoma, follicle center lymphoma, mantle cell lymphoma, Burkitt lymphoma (BL), Waldenstrom macroglobulinemia, Hodgkin lymphoma, multiple myeloma (MM), T cell leukemia, anaplasticPATENT Attorney Docket No.081906-1470154-254010PC Client Ref. No. SF2023-176 large cell lymphoma, peripheral T cell lymphoma, cutaneous T cell lymphoma, extranodal natural killer (NK) / T cell lymphoma, Epstein-Barr virus associated T cell lymphoma, T-cell acute lymphoblastic leukemia, renal cell carcinoma, nasopharyngeal carcinoma, glioblastoma, melanoma, lung carcinoma, cervix carcinoma, breast carcinoma, ovarian carcinoma, mesothelioma, metastatic cancers, lung carcinoma, pancreatic carcinoma, or osteosarcoma. In some embodiments, the autoimmune disease is systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), inflammatory bowel disease, multiple sclerosis, Sjoren’s syndrome, or systemic sclerosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 shows a schematic of an exemplary CD70-targeting chimeric antigen receptor (CAR). The CAR framework includes an CD70-targeting (or “anti-CD70”) binding domain (which is the target-binding domain of the CAR), an IgG4 hinge region with EQ mutations, a CD28 transmembrane domain, a co-stimulation domain, and a CD3 zeta (CD3ζ) cytosolic or activation domain. Typically, the transmembrane domain is embedded in the phospholipid bilayer of the cell. EQ represents the L235E and N297Q mutations in the CH2 region of the IgG4 hinge. In some embodiments, the target-binding domain has the amino acid sequence of amino acids at 23-127 of CD27 (SEQ ID NO: 2).

[0019] Figures 2A-D show in vitro anti-myeloma or anti-lymphoma activity of CD70-targeting CAR-T cells (CAR-Ts). Figures 2A-C show that an exemplary CD27-based CD70-targeting CAR- T (SEQ ID NO: 17) performed as well as or better than scFv-based anti-CD70 CAR-T (Binder 3) and anti-BCMA CAR-T positive control when tested against 3 different multiple myeloma (MM) cell lines. Figure 2D shows in vitro anti-lymphoma activity of CD70-targeting CAR-Ts when tested against HuT 78 lymphoma cell line. CD27-based CD70-targeting CAR-T (SEQ ID NO: 17; CD27 based) achieves 100% cytotoxicity at 1:1 ET ratio. Empty CAR-T (Empty.EQ.28z) and anti- BCMA CAR-T negative control (BCMA-VHH.CD8.bbz) are shown for comparison. E:T ratio = Effector:Tumor Ratio. The cell lines used are as follows: AMO1 (Figures 2A), MM.1S (Figure 2B), RPMI-8226 (Figure 2C), and Hut78 (Figure 2D).PATENT Attorney Docket No.081906-1470154-254010PC Client Ref. No. SF2023-176

[0020] Figures 3A-B show that exemplary, truncated CD27-based CD70-targeting CAR-Ts, T1- T6, did not demonstrate cytotoxicity in leukemia and MM cell lines. The “empty CAR” control is a CAR-T without an extracellular binding domain. “WT CD27” is SEQ ID NO: 17, which comprises SEQ ID NO: 2, i.e., a fragment of WT CD27. The cell lines used are as follows: NOMO- 1 (Figure 3A) and AMO1 (Figure 3B). n=2; error bars represent the standard error of the mean. E:T ratio = Effector:Tumor Ratio. Data shown is normalized to “empty CAR.”

[0021] Figure 4 shows expansion of CD70-targeting CAR-Ts in vivo. The CD27-based CD70- targeting CAR-T of the present disclosure that is based on CD70’s natural ligand, CD27. Figure 4 shows quantitative analysis of the number of CAR-T cells per microliter of murine peripheral blood for each treatment group. Data shown are the mean + / - standard deviation (SD) of 5 animals per treatment group. CD27-based CAR-T expansion (SEQ ID NO: 17) was compared to other CD70 scFv-based binders (“scFv 2,” “scFv 3,” and “scFv 4”) and B-cell maturation antigen (BCMA) CAR-T (“BCMA CAR”) on day 24 by ANOVA; **** p<0.0001.

[0022] Figures 5A-D show improved efficacy of engineered CD27 variants in CAR-T when tested against different cancer cell lines. Percent tumor lysis for different effector:tumor ratios are shown for Donor 9 CAR-T cells against MOLM-14 acute myeloid leukemia cell line (Figure 5A), Donor 10 CAR-T cells against MOLM-14 acute myeloid leukemia cell line (Figure 5B), Donor 9 CAR-T cells against RPMI-8226 multiple myeloma cell line (Figure 5C), and Donor 10 CAR-T cells against RPMI-8226 multiple myeloma cell line (Figure 5D). WT CD27 fragment: SEQ ID NO: 12. N88A: SEQ ID NO: 22. N88A, K115A, E119A (3x Ala): SEQ ID NO: 23. MPN 945: SEQ ID NO: 24.

[0023] Figures 6A-6E show the efficacy of select engineered CD27-based CAR-T variants in an in vivo mouse acute myelogenous leukemia (AML) cell line xenograft model. Tumor burden was measured twice weekly for the duration of the study, with data shown for n = 8 mice per experimental group. Figure 6A: untransduced T cells. Figure 6B: T cells transduced with wildtype (WT) CD27 CAR-T (SEQ ID NO: 12). Figure 6C: T cells transduced with the N88A CD27-based CAR-T variant (SEQ ID NO: 22). Figure 6D: T cells transduced with the 3x Ala CD27-based CAR-T variant (SEQ ID NO: 23). Figure 6E: T cells transduced with the MPN 945 CD27-based CAR-T variant (SEQ ID NO: 24).PATENT Attorney Docket No.081906-1470154-254010PC Client Ref. No. SF2023-176 DEFINITIONS

[0024] The terms “polynucleotide” and “nucleic acid” are used interchangeably to refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides. The terms include RNA, DNA, and synthetic forms and mixed polymers of the above. In particular embodiments, a nucleotide refers to a ribonucleotide, deoxynucleotide or a modified form or analog of either type of nucleotide, and combinations thereof. A reference to a nucleic acid sequence encompasses its complement unless otherwise specified. Thus, a reference to a nucleic acid molecule having a particular sequence should be understood to encompass its complementary strand, with its complementary sequence. Reference to a “polynucleotide” or “nucleic acid” that encodes a polypeptide sequence also includes codon-optimized nucleic acids and nucleic acids that comprise alternative codons that encode the same polypeptide sequence.

[0025] “Polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. As used herein, the terms encompass amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds.

[0026] The phrase “percent identical,” “percent identity,” or equivalents used in the context of two nucleic acids or polypeptides, refers to a sequence that has at least a specified level of identity, e.g., at least 50% sequence identity with a reference sequence (e.g., any SEQ ID NO included herein). Alternatively, percent identity can be any integer from 50% to 100%. Some embodiments include at least: 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, compared to a reference sequence using the programs described herein, e.g., BLAST using standard parameters, as described below.

[0027] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculatesPATENT Attorney Docket No.081906-1470154-254010PC Client Ref. No. SF2023-176 the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0028] A “comparison window,” as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection.

[0029] Algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) web site. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulationPATENT Attorney Docket No.081906-1470154-254010PC Client Ref. No. SF2023-176 of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M=1, N=-2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0030] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.01, more preferably less than about 10-5, and most preferably less than about 10-20.

[0031] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0032] As used herein the phrase “heterologous” refers to what is not found in nature. The term "heterologous sequence" refers to a sequence not normally found in a given cell in nature. As such, a heterologous nucleotide or protein sequence may be: (a) foreign to its host cell (i.e., is exogenous to the cell); (b) naturally found in the host cell (i.e., endogenous) but present at an unnatural quantity in the cell (i.e., greater or lesser quantity than naturally found in the host cell); or (c) be naturally found in the host cell but positioned outside of its natural locus.PATENT Attorney Docket No.081906-1470154-254010PC Client Ref. No. SF2023-176

[0033] The term “recombinant” or “engineered” when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.

[0034] As used herein, the term “specifically binds” to a target, e.g., human CD70, refers to a binding reaction whereby the target-binding domain of a chimeric antigen receptor (CAR) or a CD27 variant disclosed herein binds to the target with greater affinity, greater avidity, and / or greater duration than it binds to a different target. In some embodiments, a target-binding protein has at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 25-fold, 50-fold, 100-fold, 1,000-fold, 10,000-fold, or greater affinity for the target compared to an unrelated target when assayed under the same binding affinity assay conditions. The term “specific binding,” “specifically binds to,” or “is specific for” a particular target, as used herein, can be exhibited, for example, by a molecule (e.g., a ligand) having an equilibrium dissociation constant, KD, for the target of, e.g., 10-2M or smaller, e.g., 10-3M, 10-4M, 10-5M, 10-6M, 10-7M, 10-8M, 10-9M, 10-10M, 10-11M, or 10-12M. In some embodiments, a target-binding domain has a KD of less than 100 nM or less than 10 nM.

[0035] As used herein, the phrase “T cell” refers to a lymphoid cell that expresses a T cell receptor molecule. T cells include, but are not limited to, naïve T cells, stimulated T cells, primary T cells (e.g., uncultured), cultured T cells, immortalized T cells, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, combinations thereof, or sub-populations thereof. T cells can be CD4+, CD8+, or CD4+and CD8+. T cells can be helper cells, for example helper cells of type Th1, Th2, Th3, Th9, Th17, or TFH. T cells can be cytotoxic T cells. Regulatory T cells can be FOXP3+or FOXP3-. T cells can be alpha / Beta T cells or gamma / delta T cells. In some cases, the T cell is a CD4+CD25hiCD127loregulatory T cell. In some cases, the T cell is a regulatory T cell selected from the group consisting of Tr1, Th3, CD8+CD28-, Treg17, and Qa-1 restricted T cells, or a combination or sub-population thereof. In some cases, the T cell is a FOXP3+PATENT Attorney Docket No.081906-1470154-254010PC Client Ref. No. SF2023-176 T cell. In some cases, the T cell is a CD4+CD25loCD127hieffector T cell. In some cases, the T cell is a CD4+CD25loCD127hiCD45RAhiCD45RO- naïve T cell.

[0036] A T cell can be a recombinant T cell that has been genetically manipulated. In some cases, the T cell receptor is a chimeric antigen receptor (CAR) containing a target-binding domain (e.g., a natural ligand of a target), a transmembrane domain, and an intracellular / endodomain / cytosolic domain. The cytosolic domain can contain one or more signaling domains and / or adaptor domains to provide robust T cell activation and anti-antigen activity.

[0037] In some cases, the recombinant T cell has a recombinant (e.g., mutated or heterologous) T cell receptor or a chimeric antigen receptor (CAR). For example, the T cell can have a CAR with one or more mutations to alter binding specificity or signaling. As yet another example, the T cell receptor can be replaced with a polypeptide having a different binding domain or receptor domain (e.g., a CD27 fragment of variant thereof a described herein), such as a ligand, an antibody, or an antibody fragment.

[0038] As used herein, the term “antibody” means an isolated or recombinant binding agent that comprises the necessary variable region sequences or antigen-binding region to specifically bind an antigenic epitope. Therefore, an “antibody” as used herein is any form of antibody of any class or subclass or fragment thereof that exhibits the desired biological activity, e.g., binding a specific target antigen. Thus, it is used in the broadest sense and includes, but is not limited to, a monoclonal antibody (including full-length monoclonal antibodies), human antibodies, chimeric antibodies, single domain antibodies, such as nanobodies, diabodies, camelid-derived antibodies, monovalent antibodies, bivalent antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments including, but not limited to scFv, Fab, and the like so long as they exhibit the desired biological activity.

[0039] The “class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4. The antibodies described herein can be of any of these classes or subclasses.PATENT Attorney Docket No.081906-1470154-254010PC Client Ref. No. SF2023-176

[0040] The term “bispecific antibody” as used herein, refers to an antibody that binds to two or more different epitopes. In some embodiments, a bispecific antibody binds to epitopes for two different target antigens. In some embodiments, a bispecific antibody binds to two different epitopes for the same target antigen. Bispecific antibodies can be made in a number of ways. See, e.g., Brinkmann U, Kontermann RE. The making of bispecific antibodies. mAbs 2017; 9:182-212. In some embodiments, the bispecific antibodies described herein are diabodies or knob-in-a-hole IgG antibodies or otherwise use knob-in-a-hole technology. See, e.g., Xu, et al., MAbs 7(1):231- 42 (2015).

[0041] As used herein, the term “pharmaceutically acceptable carrier” refers to an excipient or diluent in a pharmaceutical composition. The pharmaceutically acceptable carrier must be compatible with the other ingredients of the formulation and not deleterious to the recipient. In the present disclosure, the pharmaceutically acceptable carrier must provide adequate pharmaceutical stability to the active ingredient. The nature of the carrier differs with the mode of administration. For example, for intravenous administration, an aqueous solution carrier is generally used; for oral administration, a solid carrier is preferred.

[0042] The terms “patient,” “subject,” “individual,” and the like are used interchangeably herein, and refer to any animal, e.g., a mammal, such as a primate. In certain non-limiting embodiments, the patient, subject or individual is a human.

[0043] The terms “treat” and “treatment” refer to both therapeutic treatment and prophylactic or preventive measures, wherein the object is to prevent or slow down an undesired physiological change or disorder. For purpose of this disclosure, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. In other embodiments the terms “treat”, “treatment” and “treating” refer to the inhibition of the progression of a proliferative disorder, either physically by, e.g., stabilization of a discernible symptom, physiologically by, e.g., stabilization of a physical parameter, or both. In otherPATENT Attorney Docket No.081906-1470154-254010PC Client Ref. No. SF2023-176 embodiments the terms “treat”, “treatment” and “treating” refer to the reduction or stabilization of tumor size or cancerous cell count.

[0044] The term “effective amount” as used herein, refers to the amount of CAR-expressing cell or CD27 variant of the present disclosure that is sufficient to effect treatment, prognosis, or diagnosis of a disease associated with CD70-expressing cells (e.g., a CD70-positive cancer cell or a CD70-positive CD4 lymphocyte), as described herein, when administered to a subject. A therapeutically effective amount will vary depending upon the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. Dosage regiments may be adjusted to provide the optimum therapeutic response. An effective amount is also one in which any toxic or detrimental effects (i.e., side effects) the CAR-expressing cell or the antibody are minimized and / or outweighed by the beneficial effects. In determining the effective amount of cells expressing the CARs or the antibodies to be administered, a physician may evaluate circulating plasma levels of immune cells or antibodies in the body and CAR or antibody-related toxicity.

[0045] As used in herein, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a CAR-T” optionally includes a combination of two or more such molecules, and the like.

[0046] The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. For example, for KDand IC50values ± 20%, ± 10%, or ± 5%, are within the intended meaning of the recited value. DETAILED DESCRIPTION I. Introduction

[0047] The present disclosure provides materials and methods related to CD27 fragments and variants thereof with improved binding to CD70. In some embodiments, CD70-targeting CAR-Ts are provided that utilize CD70’s natural ligand, CD27 and fragments and mutants thereof, for targeting CD70-expressing cells. Disclosed herein are CD27 variants (including fragments andPATENT Attorney Docket No.081906-1470154-254010PC Client Ref. No. SF2023-176 mutants) and chimeric antigen receptors (CARs) that can bind to CD70-expressing cells. CD70 is upregulated in many diseases, including, for example, hematologic and solid tumors and CD4 lymphocytes of autoimmune diseases, but minimally expressed on normal tissue. The CD70- targeting CARs and related CAR T-cell therapies (CAR-Ts) disclosed herein may be used to treat patients with a disease that is associated with CD70-positive cells. For example, no current CAR- T therapies exist for patients with CD70+ solid tumors or CD70+ blood cancer patients with acquired resistance to CD19-or BCMA-directed CAR-T therapies and relapsed or refractory AML. The CARs of the present disclosure may be introduced into immune cells, e.g., T cells, for binding to cells implicated in cancer, autoimmune diseases, or other diseases that express CD70, and to dysregulate CD70-CD27 signaling in the disease.

[0048] The CD70-targeting CAR-Ts of the present disclosure are predicted to incur fewer and less-severe side effects as existing CAR-Ts currently available. Current CAR-T therapies can cause undesirable side effects in a patient. In the case of anti-CD19 CAR-Ts, CD19 is a pan B cell marker expressed on all B cells. Thus, a side-effect of anti-CD19 CAR-Ts is the elimination of all CD19 bearing cells (both cancerous and normal B-cells), including memory B cells (which are CD19+ CD70-low) and the subsequent requirement for replacement antibody therapy (intravenous immunoglobulin or IVIG) to prevent life-threatening infections and support long-term patient survival after CAR-T treatment.

[0049] CD70, in contrast to CD19, is not expressed at high levels on normal hematopoietic tissues. CD70 is only transiently expressed on certain cell subsets of cells such as activated T cells, B cells, and dendritic cells. Though CD70 is required for full function of these cells, the level of expression of CD70 on these normal tissues is ~20 fold lower than that expressed on malignant tissues, providing an ample therapeutic window and greater likelihood of fewer on-target, off- tumor toxicities. Other CD70-targeting CAR-Ts are under current clinical development for both renal cell carcinoma in the United States (ClinicalTrials.gov ID: NCT04696731) as well as hematologic malignancies in China (ClinicalTrials.gov ID: NCT04662294). Per a review of patent literature, most of these CD70 CAR-Ts appear to use scFvs. See, e.g., Sauer, Tim et al. “CD70- specific CAR T cells have potent activity against acute myeloid leukemia without HSC toxicity.” Blood vol.138,4 (2021): 318-330. doi:10.1182 / blood.2020008221.PATENT Attorney Docket No.081906-1470154-254010PC Client Ref. No. SF2023-176

[0050] Certain fragments of CD27 have been discovered that retain CD70-binding activity without comprising the full-length wildtype CD27 sequence. The inventors have discovered, for example, that a fragment of CD27 from positions 23 to 127 of wildtype CD27 (SEQ ID NO: 1) retains CD70 binding, whereas a fragment having only positions 46-124 did not bind CD70. Thus, a fragment of CD27 starting at positions 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, or 46 (as determined relative to wildtype CD27 SEQ ID NO: 1) and ending at positions 121, 122, 123, 124, 125, 126 or 127 (but if starting at position 46, ending at position 125 or greater) can be used as a CD70-binding protein. Such CD-70 binding proteins can be fused with heterologous (non-CD27 sequences) as described herein.

[0051] In some embodiments, the CD27 variant is a truncated version of wildtype CD27 (SEQ ID NO: 1), wherein the CD27 variant comprises a portion of the wildtype CD27 sequence (SEQ ID NO: 1) and is lacking certain sequences from the N-terminus and / or the C-terminus of wildtype CD27 (SEQ ID NO: 1). While the truncations from the N-terminus and the C-terminus may vary in length and in combination, as discussed above not all truncated versions of CD27 can bind to CD70. In some embodiments, any number from 1 to 45 amino acids may be removed from the N- terminus of wildtype CD27 (SEQ ID NO: 1). In some embodiments, any number from 1 to 139 amino acids may be removed from the C-terminus of wildtype CD27 (SEQ ID NO: 1). In some embodiments, the CD27 fragment comprises the sequence of SEQ ID NO: 2, which comprises amino acids 23 to 127 of wildtype CD27 (SEQ ID NO: 1). In some embodiments, the CD27 fragment contains the cysteine-rich domain 1 (CDR-1; amino acids 1-43 of CD27) of wildtype CD27 (SEQ ID NO: 1) or a variation thereof. In some embodiments, the CD27 fragment further comprises cysteine-rich domain 2 (CDR-2; amino acids 44-85 of CD27) or a variation thereof, and cysteine-rich domain 3 (CDR-3; amino acids 86-101 of CD27) of wildtype CD27 (SEQ ID NO: 1) or a variation thereof.

[0052] In addition, certain substitutions in the CD27 wildtype sequence or fragments thereof as described herein (e.g., see below) were found to bind to CD70, or bind with enhanced affinity compared to corresponding wildtype fragments.

[0053] The CD27 variants, including fragments and mutants, described herein can be used as desired to bind to or target linked molecules to CD70 or CD70-expressing cells. In somePATENT Attorney Docket No.081906-1470154-254010PC Client Ref. No. SF2023-176 embodiments, for example, the CD27 variants described herein can be fused to a transmembrane domain such that the polypeptide is anchored to the surface of a cell, allowing for targeting of the cell to CD70-expressing cells. In some embodiments, the transmembrane domain is a CD27 transmembrane domain or a heterologous transmembrane domain. In some embodiments, the CD27 fragments and variants can form a targeting domain or a chimeric antigen receptor, and thus be linked, for example, to a transmembrane domain and an intracellular signaling domain. In some embodiments, the CD70-targeting CARs of the present disclosure have enhanced cytotoxic activity against cancer cells and / or CD70+ CD4 lymphocytes associated with autoimmune diseases when compared to CD70-targeting CARs that comprise wildtype CD27 and scFv-based anti-CD70 CARs. In some embodiments, the CD70-targeting CARs of the present disclosure may be used to treat or detect certain cancers or autoimmune diseases. In some embodiments, the CD70- targeting CARs of the present disclosure have altered binding affinities for CD70 as compared to CD70-targeting CARs that comprise wildtype CD27. In some embodiments, the CD70-targeting CARs have decreased toxicity against healthy or normal cells. II. CD27 Variants

[0054] CD27 variants that bind to CD70 are disclosed herein. Variants can be fragments (or truncations) of wildtype CD27, or can have at least one substitution compared to wildtype CD27 or a fragment thereof, wherein the substitution corresponds to at least one substitution described herein. Variants can also be fragments of wildtype CD27 with at least one substitution compared wildtype CD27 or a fragment thereof. The CD27 variants may be expressed as a recombinant protein on its own or fused to any (e.g., heterologous) polypeptide. The CD27 variants may be used in CARs, CAR-Ts, or antibodies of the present disclosure.

[0055] In some embodiments, the CD27 variant is a variant of wildtype human CD27 (SEQ ID NO: 1). In some embodiments, the CD27 variant is a truncated variant of SEQ ID NO: 1 whereby the first and / or last 1-5, 6-10, 11-15, 16-20, 21-25, 26-30, 31-35, 36-40, 41-45, 46-50, 51-55, 56- 60, 61-65, 66-70, 71-75, 76-80, 81-85, 86-70, 71-75, 76-80, 81-85, 86-70, 71-75, 76-80, 81-85, 86-90, 91-95, 96-100, 101-105, 106-110, 111-115, 116-120, 121-125, 126-130, 131-135, or 136- 140 amino acids of SEQ ID NO: 1 are absent. In some embodiments, the CD27 variant comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 1. In some embodiments, thePATENT Attorney Docket No.081906-1470154-254010PC Client Ref. No. SF2023-176 CD27 variant comprises an amino acid sequence that is at least 90, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 1, and comprises at least one amino acid substitution compared to wildtype CD27.

[0056] In some embodiments, the CD27 variant comprises an amino acid sequence that is at least 90, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 2. In some embodiments, the CD27 variant comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 2, and comprises at least one amino acid substitution. In some embodiments, the CD27 variant may also comprise a truncation whereby the first and / or last 1-5, 6-10, 11-15, 16-20, 21-25, 26-30, 31-35, or 36-40 amino acids of SEQ ID NO: 2 are absent. a. Fragments

[0057] In some embodiments, the CD27 variant is a truncated version or a fragment of wildtype CD27 (SEQ ID NO: 1). In some embodiments, the CD27 variant comprises a portion of the wildtype CD27 sequence (SEQ ID NO: 1) and is lacking certain sequences from the N-terminus and / or the C-terminus of wildtype CD27 (SEQ ID NO: 1). In some embodiments, the CD27 variant comprises a portion of the wildtype CD27 sequence (SEQ ID NO: 1) and is lacking 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 20, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acids that correspond to the amino acids on the N-terminus of SEQ ID NO: 1. In some embodiments, the CD27 variant comprises a portion of the wildtype CD27 sequence (SEQ ID NO: 1) and is lacking 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 20, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, or 139 amino acids that correspond to the amino acids on the C-terminus of SEQ ID NO: 1. In some embodiments, the CD27 variant comprises a portion of the wildtype CD27 sequence (SEQ ID NO: 1); is lacking 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 20, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acids that correspond to the amino acids on the N-terminus of SEQ ID NO: 1; and is lacking 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,PATENT Attorney Docket No.081906-1470154-254010PC Client Ref. No. SF2023-176 25, 26, 27, 28, 29, 20, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, or 139 amino acids that correspond to the amino acids on the C-terminus of SEQ ID NO: 1.

[0058] In some embodiments, the CD27 variant comprises the sequence of SEQ ID NO: 2, which is the sequence of amino acids 23 to 127 of wildtype CD27 (SEQ ID NO: 1). In some embodiments, the CD27 variant comprises a portion of SEQ ID NO: 2, and is lacking sequences from the N-terminus and / or C-terminus of SEQ ID NO: 2 to produce even shorter variants of CD27. In some embodiments, the CD27 variant comprises a portion of SEQ ID NO: 2 and is lacking 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 amino acids that correspond to the amino acids on the N-terminus of SEQ ID NO: 2. In some embodiments, the CD27 variant comprises a portion of SEQ ID NO: 2 and is lacking 1, 2, 3, 4, or 5 amino acids that correspond to the amino acids on the C-terminus of SEQ ID NO: 2. In some embodiments, the CD27 variant comprises a portion of SEQ ID NO: 2, is lacking 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 amino acids that correspond to the amino acids on the N- terminus of SEQ ID NO: 2, and is lacking 1, 2, 3, 4, or 5 amino acids that correspond to the amino acids on the C-terminus of SEQ ID NO: 2.

[0059] In some embodiments, the CD27 variant is a truncated version or a fragment of wildtype CD27 (SEQ ID NO: 1) that also comprises one or more amino acid substitutions. In some embodiments, the CD27 variant comprises a portion of the wildtype CD27 sequence (SEQ ID NO: 1), is lacking certain sequences from the N-terminus and / or the C-terminus of wildtype CD27 (SEQ ID NO: 1), and comprises one or more amino acid substitutions. In some embodiments, the CD27 variant comprises the sequence of SEQ ID NO: 2 (i.e., amino acids 23 to 127 of wildtype CD27 (SEQ ID NO: 1)) that also comprises one or more amino acid substitutions. In some embodiments, the CD27 variant comprises a portion of SEQ ID NO: 2, is lacking sequences from the N-terminus and / or C-terminus of SEQ ID NO: 2 to produce even shorter variants of CD27, and comprises one or more amino acid substitutions. Examples of amino acid substitutions are discussed below in detail.PATENT Attorney Docket No.081906-1470154-254010PC Client Ref. No. SF2023-176 b. Amino Acid Substitutions

[0060] In some embodiments, the CD27 variant comprises an amino acid substitution in one or more positions when its sequence is compared to wildtype CD27 (SEQ ID NO: 1). In some embodiments, the CD27 variant comprises an amino acid substitution as discussed in detail below in addition to one or more truncations as discussed in the preceding section.

[0061] An “amino acid substitution” refers to replacing the naturally occurring amino acid residue in a given position (e.g., the naturally occurring amino acid residue that occurs in wildtype CD27 (SEQ ID NO: 1)) with an amino acid residue other than the naturally-occurring residue. For example, the naturally occurring amino acid residue at position 88 of the wildtype CD27 polypeptide sequence (SEQ ID NO: 1) is asparagine (N88); accordingly, an amino acid substitution at N88 refers to replacing the naturally occurring asparagine with any amino acid residue other than asparagine.

[0062] An amino acid residue “corresponding to an amino acid residue [X] in [specified sequence],” or an amino acid substitution “corresponding to an amino acid substitution [X] in [specified sequence]” refers to an amino acid in a polypeptide of interest that aligns with the equivalent amino acid of a specified sequence. Generally, as described herein, the amino acid corresponding to a position of a specified CD27 polypeptide sequence can be determined using an alignment algorithm such as BLAST. In some embodiments of the present disclosure, “correspondence” of amino acid positions is determined by aligning to a region of the CD27 polypeptide comprising SEQ ID NO: 1, as discussed further herein. When a CD27 variant polypeptide sequence differs from SEQ ID NO: 1 (e.g., by changes in amino acids or addition or deletion of amino acids), it may be that a particular mutation in the CD27 variant will not be in the same position number as it is in SEQ ID NO: 1. For example, amino acid position N66 of wildtype CD27 (SEQ ID NO: 2) aligns with amino acid position N88 of CD27 variant SEQ ID NO: 1, as can be readily illustrated in an alignment of the two sequences. In this example, amino acid position 66 in SEQ ID NO: 2 corresponds to position 88 in SEQ ID NO: 1.

[0063] In some embodiments, the CD27 variant comprises an amino acid sequence with an amino acid substitution corresponding to one or more of positions 85, 88, 111, 115, 116, and 119 of SEQ ID NO: 1. In some embodiments, the CD27 variant comprises an amino acid sequencePATENT Attorney Docket No.081906-1470154-254010PC Client Ref. No. SF2023-176 comprising an amino acid substitution corresponding to position 88 of SEQ ID NO: 1 that is selected from N88A, N88C, N88D, N88E, N88F, N88G, N88H, N88I, N88K, N88L, N88M, N88P, N88Q, N88R, N88S, N88T, N88V, N88W, and N88Y. In some embodiments, the amino acid sequence comprises an amino acid substitution corresponding to position 85 of SEQ ID NO: 1 that is selected from R85A, R85C, R85D, R85E, R85F, R85G, R85H, R85I, R85K, R85L, R85M, R85N, R85P, R85Q, R85S, R85T, R85V, R85W, and R88Y. In some embodiments, the amino acid sequence comprises an amino acid substitution corresponding to position 111 of SEQ ID NO: 1 that is selected from Q111A, Q111C, Q111D, Q111E, Q111F, Q111G, Q111H, Q111I, Q111K, Q111L, Q111M, Q111N, Q111P, Q111R, Q111S, Q111T, Q111V, Q111W, and Q111Y. In some embodiments, the amino acid sequence comprises an amino acid substitution corresponding to position 115 of SEQ ID NO: 1 that is selected from K115A, K115C, K115D, K115E, K115F, K115G, K115H, K115I, K115L, K115M, K115N, K115P, K115Q, K115R, K115S, K115T, K115V, K115W, and K115Y. In some embodiments, the amino acid sequence comprises an amino acid substitution corresponding to position 116 of SEQ ID NO: 1 that is selected from E116A, E116C, E116D, E116F, E116G, E116H, E116I, E116K, E116L, E116M, E116N, E116P, E116Q, E116R, E116S, E116T, E116V, E116W, and E116Y. In some embodiments, the amino acid sequence comprises an amino acid substitution corresponding to position 119 of SEQ ID NO: 1 that is selected from E119A, E119C, E119D, E119F, E119G, E119H, E119I, E119K, E119L, E119M, E119N, E119P, E119Q, E119R, E119S, E119T, E119V, E119W, and E119Y. Any of the above substitutions can be included in any CD27 fragment described herein including but not limited to a fragment comprising or consisting of SEQ ID NO: 2 or a sequence at least 90 or 95% identical to SEQ ID NO: 2, or a portion of SEQ ID NO: 2 that is lacking 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 amino acids that correspond to the amino acids on the N-terminus of SEQ ID NO: 2, or a portion of SEQ ID NO: 2 and is lacking 1, 2, 3, 4, or 5 amino acids that correspond to the amino acids on the C- terminus of SEQ ID NO: 2, or a portion of SEQ ID NO: 2, is lacking 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 amino acids that correspond to the amino acids on the N-terminus of SEQ ID NO: 2 and lacking 1, 2, 3, 4, or 5 amino acids that correspond to the amino acids on the C-terminus of SEQ ID NO: 2.PATENT Attorney Docket No.081906-1470154-254010PC Client Ref. No. SF2023-176

[0064] In some embodiments, the CD27 variant comprises an amino acid substitution that corresponds to N88A of SEQ ID NO: 1. In some embodiments, the CD27 variant comprises amino acid substitutions that correspond to N88A, K115A, and E119A (“3x Ala” mutations) of SEQ ID NO: 1. In some embodiments, the CD27 variant comprises amino acid substitutions that correspond to Q111L and K115S (“MPN 181” mutations) of SEQ ID NO: 1. In some embodiments, the CD27 variant comprises amino acid substitutions that correspond to R85L, N88E, K115S, E116L, and E119H (“MPN 945” mutations) of SEQ ID NO: 1.

[0065] In some embodiments, the CD27 variant comprises an amino acid sequence comprising an amino acid substitution at one or more of positions 63, 66, 89, 93, 94 and 97 of SEQ ID NO: 2. In some embodiments, the CD27 variant comprises an amino acid sequence comprising an amino acid substitution at position 66 of SEQ ID NO: 2 that is selected from N66A, N66C, N66D, N66E, N66F, N66G, N66H, N66I, N66K, N66L, N66M, N66P, N66Q, N66R, N66S, N66T, N66V, N66W, and N66Y. In some embodiments, the amino acid sequence comprises an amino acid substitution at position 63 of SEQ ID NO: 2 that is selected from R63A, R63C, R63D, R63E, R63F, R63G, R63H, R63I, R63K, R63L, R63M, R63N, R63P, R63Q, R63S, R63T, R63V, R63W, and R63Y. In some embodiments, the amino acid sequence comprises an amino acid substitution at position 89 of SEQ ID NO: 2 that is selected from Q89A, Q89C, Q89D, Q89E, Q89F, Q89G, Q89H, Q89I, Q89K, Q89L, Q89M, Q89N, Q89P, Q89R, Q89S, Q89T, Q89V, Q89W, and Q89Y. In some embodiments, the amino acid sequence comprises an amino acid substitution at position 93 of SEQ ID NO: 2 that is selected from K93A, K93C, K93D, K93E, K93F, K93G, K93H, K93I, K93L, K93M, K93N, K93P, K93Q, K93R, K93S, K93T, K93V, K93W, and K93Y. In some embodiments, the amino acid sequence comprises an amino acid substitution at position 94 of SEQ ID NO: 2 that is selected from E94A, E94C, E94D, E94F, E94G, E94H, E94I, E94K, E94L, E94M, E94N, E94P, E94Q, E94R, E94S, E94T, E94V, E94W, and E94Y. In some embodiments, the amino acid sequence comprises an amino acid substitution at position 97 of SEQ ID NO: 2 that is selected from E97A, E97C, E97D, E97F, E97G, E97H, E97I, E97K, E97L, E97M, E97N, E97P, E97Q, E97R, E97S, E97T, E97V, E97W, and E97Y.

[0066] In some embodiments, the extracellular target-binding domain comprises an amino acid substitution that corresponds to N66A of SEQ ID NO: 2. In some embodiments, the extracellular target-binding domain comprises amino acid substitutions that correspond to N66A, K93A, andPATENT Attorney Docket No.081906-1470154-254010PC Client Ref. No. SF2023-176 E94A (“3x Ala” mutations) of SEQ ID NO: 2. In some embodiments, the extracellular target- binding domain comprises amino acid substitutions that correspond to Q89L and K93S (“MPN 181” mutations) of SEQ ID NO: 2. In some embodiments, the extracellular target-binding domain comprises amino acid substitutions that correspond to comprising R63L, N66E, K93S, E94L, and E97H (“MPN 945” mutations) of SEQ ID NO: 2. c. Exemplary CD27 Variants

[0067] In some embodiments, the CD27 variant comprises the amino acid sequence of SEQ ID NO: 2.

[0068] In some embodiments, the CD27 variant comprises the amino acid sequence of SEQ ID NO: 2 and an amino acid substitution that corresponds to N88A of SEQ ID NO: 1. In some embodiments, the CD27 variant comprises the amino acid sequence of SEQ ID NO: 4.

[0069] In some embodiments, the CD27 variant comprises the amino acid sequence of SEQ ID NO: 2 and amino acid substitutions that correspond to N88A, K115A, and E119A (“3x Ala” mutations) of SEQ ID NO: 1. In some embodiments, the CD27 variant comprises the amino acid sequence of SEQ ID NO: 6 (the “3x Ala” sequence).

[0070] In some embodiments, the CD27 variant comprises the amino acid sequence of SEQ ID NO: 2 and amino acid substitutions that correspond to Q111L and K115S (“MPN 181” mutations) of SEQ ID NO: 1. In some embodiments, the CD27 variant comprises the amino acid sequence of SEQ ID NO: 8 (the “MPN 181” sequence).

[0071] In some embodiments, the CD27 variant comprises the amino acid sequence of SEQ ID NO: 2 and amino acid substitutions that correspond to R85L, N88E, K115S, E116L, and E119H (“MPN 945” mutations) of SEQ ID NO: 1. In some embodiments, the CD27 variant comprises the amino acid sequence of SEQ ID NO: 10 (the “MPN 945” sequence). III. Chimeric Antigen Receptors

[0072] Chimeric antigen receptors (CARs) of the present disclosure can comprise, for example (1) an extracellular target-binding domain, (2) a transmembrane domain, (3) a hinge domain, and (4) an intracellular signaling domain. The extracellular target-binding domain comprises a polypeptide that binds to CD70. The transmembrane domain links the extracellular target-bindingPATENT Attorney Docket No.081906-1470154-254010PC Client Ref. No. SF2023-176 domain and the intracellular signaling domain, and anchors the CAR to the plasma membrane of the host cell that is modified to express the CAR, e.g., the plasma membrane of a human immune cell. The hinge domain links the extracellular target-binding domain and the transmembrane domain for positioning the extracellular target-binding domain.

[0073] A CAR construct encoding a CAR may also comprise a sequence that encodes a signal peptide to target the extracellular domain to the cell surface. a. Extracellular Target-Binding Domain

[0074] In some embodiments, the extracellular target-binding domain comprises a polypeptide that binds to CD70. In some embodiments, the extracellular target-binding domain comprises a natural ligand or a variant thereof of CD70. In some embodiments, the extracellular target-binding domain comprises a CD27 variant. In some embodiments, the extracellular target-binding domain does not comprise wildtype full-length CD27 (SEQ ID NO: 1). As discussed in detail in the preceding sections, many variations of CD27 variants as described herein may be used in the extracellular target-binding domain of a CAR.

[0075] In some embodiments, the extracellular target-binding domain comprises a portion of SEQ ID NO: 1 whereby the first and / or last 1-5, 6-10, 11-15, 16-20, 21-25, 26-30, 31-35, 36-40, 41-45, 46-50, 51-55, 56-60, 61-65, 66-70, 71-75, 76-80, 81-85, 86-70, 71-75, 76-80, 81-85, 86-70, 71-75, 76-80, 81-85, 86-90, 91-95, 96-100, 101-105, 106-110, 111-115, 116-120, 121-125, 126- 130, 131-135, or 136-140 amino acids of SEQ ID NO: 1 are absent. In some embodiments, the extracellular target-binding domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 1. In some embodiments, the extracellular target-binding domain comprises an amino acid sequence that is at least 90, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 1, and comprises at least one amino acid substitution compared to wildtype CD27.

[0076] In some embodiments, the extracellular target-binding domain comprises an amino acid sequence that is at least 90, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 2. In some embodiments, the extracellular target-binding domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 2, and comprises at least one amino acid substitution. In some embodiments, the extracellular target-binding domain may also comprise a portion of SEQ ID NO:PATENT Attorney Docket No.081906-1470154-254010PC Client Ref. No. SF2023-176 2 whereby the first and / or last 1-5, 6-10, 11-15, 16-20, 21-25, 26-30, 31-35, or 36-40 amino acids of SEQ ID NO: 2 are absent.

[0077] In some embodiments, the extracellular target-binding domain comprises an amino acid substitution that corresponds to N88A of SEQ ID NO: 1. In some embodiments, the extracellular target-binding domain comprises amino acid substitutions that correspond to N88A, K115A, and E119A (“3x Ala” mutations) of SEQ ID NO: 1. In some embodiments, the extracellular target- binding domain comprises amino acid substitutions that corresponds to Q111L and K115S (“MPN 181” mutations) of SEQ ID NO: 1. In some embodiments, the extracellular target-binding domain comprises amino acid substitutions that correspond to R85L, N88E, K115S, E116L, and E119H (“MPN 945” mutations) of SEQ ID NO: 1.

[0078] In some embodiments, the extracellular target-binding domain comprises an amino acid substitution that corresponds to N66A of SEQ ID NO: 2. In some embodiments, the extracellular target-binding domain comprises amino acid substitutions that correspond to N66A, K93A, and E94A (“3x Ala” mutations) of SEQ ID NO: 2. In some embodiments, the extracellular target- binding domain comprises amino acid substitutions that corresponds to Q89L and K93S (“MPN 181” mutations) of SEQ ID NO: 2. In some embodiments, the extracellular target-binding domain comprises amino acid substitutions that correspond to comprising R63L, N66E, K93S, E94L, and E97H (“MPN 945” mutations) of SEQ ID NO: 2.

[0079] In some embodiments, the extracellular target-binding domain comprises the amino acid sequence of SEQ ID NO: 2, 4, 6 (the “3x Ala” sequence), 8 (the “MPN 181” sequence), or 10 (the “MPN 945” sequence). b. Transmembrane Domain

[0080] Any transmembrane suitable for use in a CAR construct may be employed. A transmembrane domain incorporated into a CAR construct may be derived either from a natural, synthetic, semi-synthetic, or recombinant source. Such transmembrane domains, include, but are not limited to, all or part of the transmembrane domain of the alpha, beta or zeta chain of the T- cell receptor, CD28, CD27, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. In some embodiments, a transmembrane domain may include at least the transmembrane region(s) of, e.g., KIRDS2, OX40, CD2, CD27,PATENT Attorney Docket No.081906-1470154-254010PC Client Ref. No. SF2023-176 LFA-1 (CD 11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2R beta, IL2R gamma, IL7R a, ITGAl, VLAl, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDl la, LFA-1, ITGAM, CDl lb, ITGAX, CDl lc, ITGB 1, CD29, ITGB2, CD 18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100, (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME, (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKG2D, or NKG2C. In some embodiments, the transmembrane domain is the native CD27 transmembrane domain.

[0081] In some embodiments, the transmembrane domain is linked to a hinge domain. In some embodiments, the combined transmembrane and hinge domain comprises the sequence of SEQ ID NO: 14. c. Hinge Domain

[0082] In some embodiments, the CAR may contain one or more hinge domains that link the extracellular target-binding domain and the transmembrane domain for positioning the extracellular target-binding domain. Such a hinge domain may be derived either from a natural, synthetic, semi-synthetic, or recombinant source. The hinge domain can include the amino acid sequence of a naturally occurring immunoglobulin hinge region, e.g., a naturally occurring human immunoglobulin hinge region, or an altered immunoglobulin hinge region. Illustrative hinge domains suitable for use in the CARs described herein include the hinge region derived from the extracellular regions of type 1 membrane proteins such as CD8 alpha, CD4, CD28, PD1, CD 152, and CD7, which may be wildtype hinge regions from these molecules or may be altered.

[0083] In some embodiments, the hinge domain is based on the hinge region of a human immunoglobulin IgG1 or IgG4. In some embodiments, the hinge region includes the IgG1 or IgG4’s CH2 region, which may comprise one or more mutations. In some embodiments, the mutation is L235E, N297Q, or both L235E and N297Q (which is known as the EQ mutation in the IgG4 hinge region).PATENT Attorney Docket No.081906-1470154-254010PC Client Ref. No. SF2023-176

[0084] In some embodiments, the hinge domain is linked to a transmembrane domain. In some embodiments, the combined hinge and transmembrane domain comprise the amino acid sequence of SEQ ID NO: 14. d. Intracellular Signaling Domain

[0085] A CAR construct of the present disclosure can include one or more intracellular signaling domains, also referred to herein as co-stimulatory domains, activation domains, or cytoplasmic domains that activate or otherwise modulate an immune cell, (e.g., a T lymphocyte). The intracellular signaling domain is generally responsible for activation of at least one of the normal effector functions of the immune cell in which the CAR has been introduced. For example, a “first- generation CAR” generally has a CD3 zeta (CD3ζ) signaling domain. Additional co-stimulatory intracellular domains may also be introduced (e.g., second and third generation CARS) and further domains including homing and suicide domains may be included in CAR constructs.

[0086] In some embodiments, a co-stimulatory domain is used that increases CAR immune T cell cytokine production. In another embodiment, a co-stimulatory domain is used that facilitates immune cell (e.g., T cell) replication. In still another embodiment, a co-stimulatory domain is used that prevents CAR immune cell (e.g., T cell) exhaustion. In another embodiment, a co-stimulatory domain is used that increases immune cell (e.g., T cell) antitumor activity. In still a further embodiment, a co-stimulatory domain is used that enhances survival of CAR immune cells (e.g., T cells) (e.g., post-infusion into patients).

[0087] Examples of intracellular signaling domains for use in a CAR include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any recombinant sequence that has the same functional capability.

[0088] A primary signaling domain regulates primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way. Primary intracellular signaling domains that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine- based activation motifs or ITAMs.PATENT Attorney Docket No.081906-1470154-254010PC Client Ref. No. SF2023-176

[0089] Examples of ITAM containing primary intracellular signaling domains include those of CD3 zeta, common FcR gamma, Fc gamma Rlla, FcR beta (Fc Epsilon Rib), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12. In one embodiment, a CAR comprises an intracellular signaling domain, e.g., a primary signaling domain of CD3 zeta.

[0090] An intracellular signaling domain of a CAR can comprise a primary intracellular signaling domain only, or may comprise additional desired intracellular signaling domain(s) useful in the context of a CAR of the disclosure. For example, the intracellular signaling domain of the CAR can comprise a CD3 zeta chain portion and a co-stimulatory signaling domain. The co- stimulatory signaling domain refers to a portion of the CAR comprising the intracellular domain of a co-stimulatory molecule. A co-stimulatory molecule is a cell surface molecule other than an antigen receptor or its ligands that is required for an efficient response of lymphocytes to an antigen. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that binds to CD83, and the like. For example, CD27 costimulation has been demonstrated to enhance expansion, effector function, and survival of human CART cells in vitro and augments human T cell persistence and antitumor activity in vivo (Song et al. Blood.2012; 119(3):696-706). Further examples of such co-stimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD 160, CD 19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDl la, LFA-1, ITGAM, CDl lb, ITGAX, CDl lc, ITGB 1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), NKG2D, CEACAMl, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM, (SLAMFl, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, and CD 19a.

[0091] In some embodiments, the intracellular domain comprises a co-stimulatory domain and an intracellular CD3 zeta (CD3ζ) signaling domain. In some embodiments, the intracellular co- stimulatory domain comprises the sequence of SEQ ID NO: 15. In some embodiments, the intracellular CD3ζ signaling domain comprises the sequence of SEQ ID NO: 16.PATENT Attorney Docket No.081906-1470154-254010PC Client Ref. No. SF2023-176 e. Exemplary Chimeric Antigen Receptors

[0092] In some embodiments, the chimeric antigen receptor (CAR) comprises a CD27 variant (i.e., an amino acid sequence derived from WT, full-length human CD27 (SEQ ID NO: 1) that is capable of binding to CD70. In some embodiments, the CAR comprises a truncated CD27. In some embodiments, the CAR comprises a truncated CD27 comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 2. In some embodiments, the CAR comprises the truncated CD27 amino acid sequence of SEQ ID NO: 2. In some embodiments, the CAR comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 12. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, the CAR comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 17. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 17.

[0093] In some embodiments, the CAR comprises a CD27 variant comprising the amino acid sequence of SEQ ID NO: 2 and an amino acid substitution that corresponds to N88A of SEQ ID NO: 1. In some embodiments, the CAR comprises a CD27 variant comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 4. In some embodiments, the CAR comprises the CD27 variant amino acid sequence of SEQ ID NO: 4. In some embodiments, the CAR comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 22. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 22.

[0094] In some embodiments, the CAR comprises a CD27 variant comprising the amino acid sequence of SEQ ID NO: 2 and amino acid substitutions that correspond to N88A, K115A, and E119A (“3x Ala” mutations) of SEQ ID NO: 1. In some embodiments, the CAR comprises a CD27 variant comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 6. In some embodiments, the CAR comprises the CD27 variant amino acid sequence of SEQ ID NO: 6. In some embodiments, the CAR comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 23. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 23.

[0095] In some embodiments, the CAR comprises a CD27 variant comprising the amino acid sequence of SEQ ID NO: 2 and amino acid substitutions that correspond to R85L, N88E, K115S, E116L, and E119H (“MPN 945” mutations) of SEQ ID NO: 1. In some embodiments, the CARPATENT Attorney Docket No.081906-1470154-254010PC Client Ref. No. SF2023-176 comprises a CD27 variant comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 10. In some embodiments, the CAR comprises the CD27 variant amino acid sequence of SEQ ID NO: 10. In some embodiments, the CAR comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 24. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 24. IV. Antibodies

[0096] In some embodiments, a CD27 variant of the present disclosure may be covalently linked to an antibody. Many CD27 variants as discussed in detail in the sections above. In some embodiments, the antibody is a bispecific antibody. In some embodiments, the bispecific antibodies comprise a CD27 variant as described herein that specifically binds to CD70 and a second antigen-binding region or variable region that specifically binds to another target. In some embodiments, a bispecific antibody can be constructed by linking a CD27 variant with a Bispecific T Cell Engager (BiTE) that can bind to CD3. In some embodiments, a bispecific antibody can be constructed by linking a CD27 variant with a natural killer (NK) cell engager that can bind to CD16. V. Polynucleotides

[0097] Recombinant nucleic acids encoding any of the CD27 variants or CAR disclosed herein are provided. In some embodiments, the nucleic acid comprises the sequence of SEQ ID NO: 3. In some embodiments, the nucleic acid comprises the sequence of SEQ ID NO: 5. In some embodiments, the nucleic acid comprises the sequence of SEQ ID NO: 7. In some embodiments, the nucleic acid comprises the sequence of SEQ ID NO: 9. In some embodiments, the nucleic acid comprises the sequence of SEQ ID NO: 11.

[0098] Nucleic acids or vectors encoding the desired CD27 variant or CAR may be introduced into cells such that the cells may express the desired CD27 variant or CAR. The nucleic acids can be prepared according to standard methods known to those of skill in the art. Also, using the sequence information provided herein, the CD27 variants can be chemically synthesized using well known methods of peptide synthesis.PATENT Attorney Docket No.081906-1470154-254010PC Client Ref. No. SF2023-176

[0099] Molecular cloning techniques to achieve these ends are known in the art. A wide variety of cloning and in vitro amplification methods are suitable for the construction of recombinant nucleic acids. Examples of these techniques and instructions sufficient to direct persons of skill through many cloning exercises are found in Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Inc., San Diego, CA (Berger); Sambrook et al. (1989) Molecular Cloning – A Laboratory Manual (2nd ed.) Vol.1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY, (Sambrook); and Current Protocols in Molecular Biology, F.M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1994 Supplement) (Ausubel). Methods of producing recombinant immunoglobulins are also known in the art. See, Cabilly, U.S. Patent No.4,816,567; and Queen et al. (1989) Proc. Natl Acad. Sci. USA 86: 10029-10033.

[0100] Many methods for introducing nucleic acids and viral vectors (e.g., viral particles) into a target cell (e.g., a CD8+T cell) are known to one of ordinary skill in the art. Non-limiting examples of suitable methods include electroporation (e.g., nucleofection), viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome- mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, microparticle- or nanoparticle-mediated nucleic acid delivery, and the like. In some embodiments, a viral vector may be used, such as an adenovirus, adeno-associated virus (AAV), lentivirus vector, a vaccinia virus vector, or any of a number of different vectors. VI. Cells

[0101] Cells comprising a nucleic acid for expressing a CAR or a CD27 variant as described herein are provided. In some embodiments, the cells demonstrate enhanced cytotoxic activity against CD70-positive cancer cells and / or CD70-positive CD4 lymphocytes associated with autoimmune diseases. In some embodiments, the cells have decreased toxicity against healthy or normal cells.

[0102] The cells provided herein are human cells, though they are not limited by the type of human immune cells genetically modified to express a CAR. Illustrative immune cells include, but are not limited to, human T cells, e.g., alpha / beta T cells and gamma / delta T cells, B cells, naturalPATENT Attorney Docket No.081906-1470154-254010PC Client Ref. No. SF2023-176 killer (NK) cells, natural killer T (NKT) cells, mast cells, macrophages, monocytes, induced pluripotent stem cell, hematopoietic stem cell, myeloid progenitor cell, lymphoid progenitor cell, and myeloid-derived phagocytes. In some embodiments, the T cells are CD8+ T cells Treg cells.

[0103] In some embodiments, the cells used to treat a patient were originally obtained from the same patient; these cells are referred to as autologous cells. In some embodiments, the immune cells, e.g., T cells, are autologous cells from the patient to undergo immunotherapy. In some embodiments, the cells used to treat a patient were obtained from a different subject; these cells are referred to as allogeneic cells. In some embodiments, the immune cells are allogeneic. Methods of making CAR-expressing cells are described, e.g., in U.S. Patent Publication Nos.2016 / 0185861 and 2019 / 0000880. VII. Methods of Use

[0104] CD70 is upregulated in many diseases but minimally expressed on normal tissue. In some embodiments, a CD27 variant, a cell expressing a CD70-targeting CAR, or a bispecific antibody as disclosed herein may be used to diagnose and / or treat patients with a disease that is associated with CD70-positive cells, i.e., cells that express CD70 on its surface. In some embodiments, the CD27 variant, the CAR-expressing cells, or the bispecific antibody may block signaling in the CD27-CD70 pathway. In some embodiments, the CD27 variant, CAR-expressing cells, or the bispecific antibody may reduce disease manifestations in human patients and / or animal models of disease. a. Therapeutic Methods

[0105] A composition comprising a CD27 variant, CAR-expressing cell, or bispecific antibody of the present disclosure may be administered to a patient to treat a disease associated with elevated CD70-expressing cells. Compositions that may be used in therapeutic methods are also disclosed below.

[0106] In some embodiments, the composition comprises a human cell comprising a nucleic acid for expression of a CAR of the present disclosure. In some embodiments, the method comprises allogeneic cells, which are cells obtained from a patient who will receive the CAR- expressing cells for treatment of disease. Examples of suitable cells are disclosed above. In somePATENT Attorney Docket No.081906-1470154-254010PC Client Ref. No. SF2023-176 embodiments, the method comprises autologous cells, which are cells obtained from a subject other than the patient who will receive the CAR-expressing cells for treatment of disease. In some embodiments, the disease is cancer or an autoimmune disease. Examples of cancers and autoimmune diseases are disclosed below. b. Detection Methods

[0107] In some embodiments, the CD27 variants disclosed herein can be used to detect the presence of a CD70-expressing cell that is implicated in disease. The CD27 variants may be used for in vitro diagnostic or monitoring methods, e.g., by contacting live cells or fixed cells from a subject or a patient.

[0108] When used for detection or diagnosis, the CD27 variants is typically conjugated or otherwise associated with a detectable label. The association can be direct e.g., a covalent bond, or indirect, e.g., using a secondary binding agent, chelator, or linker. Suitable detectable labels include, but are not limited to radio-opaque labels, nanoparticles, PET labels, MRI labels, radioactive labels, and the like. Among the radionuclides and useful in various embodiments, gamma-emitters, positron-emitters, x-ray emitters and fluorescence-emitters are suitable for localization, diagnosis and / or staging, and / or therapy, while beta and alpha-emitters and electron and neutron-capturing agents, such as boron and uranium, also can be used for therapy.

[0109] In various embodiments the detectable labels can be used in conjunction with an external detector and / or an internal detector and provide a means of effectively localizing and / or visualizing cancer cells expressing CD70. Such detection / visualization can be useful in various contexts including, but not limited to pre-operative and intraoperative settings. Thus, in certain embodiments this disclosure relates to a method of intraoperatively detecting cancers that express CD70 in the body of a mammal. These methods typically involve administering to the subject a composition comprising, in a quantity sufficient for detection by a detector (e.g., a gamma detecting probe), a CD27 variant labeled with a detectable label as described herein, and, after allowing the active substance to be taken up by the target tissue, and preferably after blood clearance of the label, subjecting the subject to a radio-immunodetection technique in the relevant area of the body, e.g., by using a gamma detecting probe.PATENT Attorney Docket No.081906-1470154-254010PC Client Ref. No. SF2023-176

[0110] In certain embodiments the label-bound CD27 variants can be used in the technique of radio-guided surgery, wherein relevant tissues in the body of a subject can be detected and located intraoperatively by means of a detector, e.g., a gamma detecting probe. A surgeon can, intraoperatively, use this probe to find the tissues in which uptake of the compound labeled with a radioisotope, that is, e.g., a low-energy gamma photon emitter, has taken place. In certain embodiments such methods are particularly useful in localizing and removing secondary cancers produced by metastatic cells from a primary tumor.

[0111] The CD27 variants described herein can be coupled directly to the radio-opaque moiety (e.g., at an available cysteine) or they can be attached to a “package” (e.g., a chelate, a liposome, a polymer microbead, a nanoparticle, etc.) carrying, containing, or comprising the radio-opaque material, e.g., as described below.

[0112] In addition to radio-opaque labels, other labels are also suitable for use. Detectable labels suitable for use include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels in the include magnetic beads (e.g., DYNABEADS™), fluorescent dyes (e.g., fluorescein isothiocyanate, texas red, rhodamine, green fluorescent protein, and the like), radiolabels (e.g., H, I, S, C, or P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic (e.g. polystyrene, polypropylene, latex, etc.) beads, nanoparticles, quantum dots, and the like.

[0113] In certain embodiments, suitable radiolabels include, but are not limited to Tc, Tc , Ru, Ru, Tc, Y, Y, Zr, Y, Br, As, Br, Se, As, Ga, Ga, Ga, Ga, Cu, Cu, Cu, Cu, Cu, F e, Co, Co, Mn, Fe, Cr, Sc, H, S, P, P, Ac, Ac, Ra, Bi, Pb, Bi, At, Pb, Hg, T1, Aufor example, certain radiolabels may be detected using photographic film, scintillation detectors, PET imaging, MRI, and the like. Fluorescent markers can be detected using a photodetector to detect emitted illumination. Enzymatic labels are typically detected by providing the enzyme with aPATENT Attorney Docket No.081906-1470154-254010PC Client Ref. No. SF2023-176 substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label. c. Diseases

[0115] CD70 is a highly abundant on the surface of cells implicated in certain cancers. In some embodiments, the CD27 variants disclosed herein may be used for cancer treatment. In some embodiments, the CD27 variants facilitate tumor detection and / or localization. In some embodiments, the cancer is a blood cancer or leukemia. In some embodiments, the leukemia is acute myelogenous leukemia (AML), B-cell acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL or B-CLL), hairy cell leukemia, B-cell prolymphocytic leukemia, Non-Hodgkin lymphoma or Non-Hodgkin’s lymphoma, Hodgkin lymphoma or Hodgkin’s lymphoma, multiple myeloma (MM), or T-cell leukemia. In some embodiments, the non-Hodgkin lymphoma is diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, follicle center lymphoma, mantle cell lymphoma, Burkitt lymphoma (BL), orWaldenstrom macroglobulinemia. In some embodiments, the T-cell leukemia is anaplastic large cell lymphoma, peripheral T cell lymphoma, cutaneous T cell lymphoma, extranodal natural killer (NK) / T-cell lymphoma, Epstein- Barr virus associated T cell lymphoma, or T-cell acute lymphoblastic leukemia.

[0116] In some embodiments, the cancer is a solid tumor. In some embodiments, the tumor is renal cell carcinoma, nasopharyngeal carcinoma, glioblastoma, melanoma, lung carcinoma, cervix carcinoma, breast carcinoma, ovarian carcinoma, mesothelioma, or a metastatic cancer. In some embodiments, the metastatic cancer is lung carcinoma, pancreatic carcinoma, or osteosarcoma.

[0117] CD70-expressing CD4 lymphocytes are elevated in certain autoimmune disease. In some embodiments, the CD27 variants of the present disclosure may be used as a method of detection or treatment for an autoimmune disease. In some embodiments, the autoimmune disease is systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), inflammatory bowel disease, multiple sclerosis, Sjogren’s syndrome, or systemic sclerosis. VIII. Compositions

[0118] Pharmaceutical compositions comprising a CD27 variant, a bispecific antibody, or a CAR-expressing cell that binds to CD70-expressing cells as described herein can include one orPATENT Attorney Docket No.081906-1470154-254010PC Client Ref. No. SF2023-176 more pharmaceutically acceptable carriers. Acceptable carriers and excipients in the pharmaceutical compositions are nontoxic to recipients at the dosages and concentrations employed. Acceptable carriers and excipients may include buffers, antioxidants, preservatives, polymers, amino acids, and carbohydrates. Pharmaceutical compositions may be administered parenterally in the form of an injectable formulation. Pharmaceutical compositions for injection (i.e., intravenous injection) can be formulated using a sterile solution or any pharmaceutically acceptable liquid as a vehicle. Pharmaceutically acceptable vehicles include, but are not limited to, sterile water, physiological saline, and cell culture media (e.g., Dulbecco’s Modified Eagle Medium (DMEM), α-Modified Eagles Medium (α-MEM), F-12 medium). Formulation methods are known in the art, see e.g., Banga (ed.) Therapeutic Peptides and Proteins: Formulation, Processing and Delivery Systems (2nd ed.) Taylor & Francis Group, CRC Press (2006).

[0119] The pharmaceutical composition may be formed in a unit dose form as needed. The effective amount of active component, e.g., an antibody, a CD27 variant, or a CAR-expressing cell as described herein, included in the pharmaceutical preparations is such that a suitable dose within the designated range is provided. For example, a dose for an antibody or a CD27 variant may be within the range of 0.01-500 mg / kg of body weight.

[0120] Pharmaceutical compositions described herein may be formulated for subcutaneous administration, intramuscular administration, intravenous administration, parenteral administration, intra-arterial administration, intrathecal administration, or intraperitoneal administration. The pharmaceutical composition may also be formulated for, or administered via, oral, nasal, spray, aerosol, rectal, or vaginal administration. For injectable formulations, various effective pharmaceutical carriers are known in the art. In some embodiments, pharmaceutical compositions may be administered locally or systemically (e.g., locally). In particular embodiments, pharmaceutical compositions may be administered locally at the affected area, such as skin or cancerous tissue.

[0121] The dosage of the pharmaceutical compositions depends on factors including the route of administration, the disease to be treated, and physical characteristics, e.g., age, weight, general health, of the subject. In some embodiments, the amount of active ingredient (e.g., an antibody, a CD27 variant, or a CAR-expressing cell as described herein) contained within a single dose arePATENT Attorney Docket No.081906-1470154-254010PC Client Ref. No. SF2023-176 administered in an amount that effectively prevents, delays, or treats the disease without inducing significant toxicity. The dosage may be adapted by the physician in accordance with conventional factors such as the extent of the disease and different parameters of the subject.

[0122] The pharmaceutical compositions may be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective to result in an improvement or remediation of the symptoms. The pharmaceutical compositions may be administered in a variety of dosage forms, e.g., subcutaneous dosage forms, intravenous dosage forms, and oral dosage forms (e.g., ingestible solutions, drug release capsules). Pharmaceutical compositions containing the active ingredient (e.g., an antibody, a CD27 variant, or a cell expressing CARs as described herein) may be administered to a subject in need thereof, for example, one or more times (e.g., 1-10 times or more) daily, weekly, monthly, biannually, annually, or as medically necessary. Dosages may be provided in either a single or multiple dosage regimens. The timing between administrations may decrease as the medical condition improves or increase as the health of the patient declines. EXAMPLES Overview

[0123] The following Examples relate to targeting CD70 with CAR-Ts, which comprise a portion of CD27 (SEQ ID NO: 2) that is incorporated into a second-generation CAR-T backbone (Figure 1). The Examples demonstrate selectivity and efficacy of exemplary CD70-targeting CAR- Ts in targeting and eliminating MM, lymphoma, and leukemia cells in vitro (Figures 2A-D and 3A-B). The exemplary CAR-Ts were modified by the incorporation of certain amino acid substitutions as described herein in the CD27 portion for altered binding affinities to CD70 and improved cytotoxicity against CD70+ tumors (as compared to wildtype CD27) (Figures 5A-D). Example 1 – Materials and Methods

[0124] CD70-positive cell lines. Multiple myeloma (MM) cell lines that were used were KHM11, KMS26, LP1, AMO1, MM1.S, LP1, and RPMI8226. A leukemia cell line that was used was NOMO-1. A lymphoma cell line that was used was HuT 78. Lentiviral transduction methods were used to stably express luciferase in these cell-lines.PATENT Attorney Docket No.081906-1470154-254010PC Client Ref. No. SF2023-176

[0125] Fragment of CD27 extracellular domain. CD27 variants were designed based on the human CD70:CD27 co-crystal structure (PDB: 7KX0). Several variants were designed to determine a minimal fragment of CD27 that can serve as an CD70-targeting binder in our CAR-T framework. A fragment from alanine 23 (Ala23) to serine 127 (Ser127) of wildtype CD27 sequence was cloned into a lentiviral-based CAR-T framework. This CD27 fragment from Ala23 to Ser127 was tested for cytotoxicity in in vitro models against CD70+ human tumor cell lines.

[0126] CD27 mutants. Key amino acid interactions between CD27 and CD70 were identified based on the human CD70:CD27 co-crystal structure (PDB: 7KX0). Key CD27 amino acids were mutated in different combinations and the CD70:CD27 protein:protein interface was modeled using Rosetta. The mutants were analyzed for increased binding affinity between CD27 and CD70. Several CD27 mutants that were predicted to increase binding affinity of CD27 to CD70; these mutants each had a decrease in Rosetta energy units ranging from -126.6 to -135.5 vs. wildtype. In addition, variants with lower affinity were designed by mutating a subset of key amino acids on CD27 to alanines, thereby abrogating their sidechain interactions.

[0127] Chimeric antigen receptor (CAR) backbone. The CD27-based CARs comprised a CD27 fragment (i.e., a truncation of wildtype CD27) or mutant, an IgG4 hinge domain, a CD28 transmembrane region, a CD28 co-stimulatory domain, and an intracellular CD3ζ signaling domain (Figure 1). The lentiviral or adeno-associated viral (AAV) CAR framework for each of these constructs also comprised a CD8α signal peptide that is cleaved away to produce the final CAR protein.

[0128] Two CAR frameworks are described in these Examples. The first CAR framework is a lentiviral framework and is used in the WT CD27-CAR-2 sequence (SEQ ID NO: 17) of Examples 2-5 below. The WT CD27-CAR-2 sequence is compatible with lentiviral transduction, which is the same method used in all currently FDA-approved CAR-T therapies. See, e.g., Labbé, Roman P et al. “Lentiviral Vectors for T Cell Engineering: Clinical Applications, Bioprocessing and Future Perspectives.” Viruses vol.13,81528.2 Aug.2021.

[0129] The second CAR framework is an AAV-compatible framework and is used in the WT CD27-CAR-1 sequence (SEQ ID NO: 12) of Examples 6-8 below. The WT CD27-CAR-2 sequence is compatible with homology-directed repair-driven expression of the CAR at the TRACPATENT Attorney Docket No.081906-1470154-254010PC Client Ref. No. SF2023-176 locus. See, e.g., Eyquem, Justin et al. “Targeting a CAR to the TRAC locus with CRISPR / Cas9 enhances tumour rejection.” Nature vol. 543,7643 (2017): 113-117. This method is useful for efficient targeting of the TRAC locus and for screening CAR mutations, as well as effective CAR internalization and re-expression.

[0130] CD27-based CAR-T. To evaluate the efficacy of the CD27-mutant CARs, a TRAC knock-in CAR system was used in Examples 6-8 below. Each CAR construct was introduced into T cells at the TRAC locus of the TCRα gene. An AAV vector was used to deliver a homology- directed repair template containing the CAR to the T cells. The cells were electroporated with a CRISPR-Cas9 system which makes a double-stranded cut at exon 1 of TRAC. The CAR itself consists of a CD8α signal peptide, followed by the extracellular CD27-based binding domain, a CD28 hinge and transmembrane region, a CD28 co-stimulatory domain, and an intracellular CD3ζ signaling domain. In Example 5 below, CAR-T lentiviral transduction was performed on paired CD4 / CD8 human T cells from at least 3 healthy donors.

[0131] In vitro cytotoxicity assays. Effector function of CD70-targeting CAR-Ts was assessed by cytotoxicity against CD70-positive cell lines using a 24-hour luciferase-based tumor killing assay.

[0132] Flow cytometry. MM cell lines were profiled by flow cytometry.

[0133] Patient samples. Primary patient samples were obtained under protocols approved by the institutional review board (IRB).

[0134] In vivo studies. Mice bearing MM.1S tumors were used. MM.1S tumor cells were inoculated into NOD scid gamma (NSG) mice intravenously at a dose of 1e6 tumor cells / mouse at Day -7. At Day 7, 5e6 CAR-T cells were administered intravenously. In vivo efficiency of CAR- Ts was assessed using bioluminescence.

[0135] NSG mice bearing MOLM-14 acute myelogenous leukemia (AML) cell line tumors were also used. Mice were injected with 1 million luciferase-expressing MOLM-14 cells intravenously, followed by 500,000 CAR-expressing T-cells 4 days later. Tumor burden was assessed by intraperitoneal injection of D-luciferin, followed by bioluminescence imaging (BLI) to quantify the total flux produced by luciferase-expressing tumor cells in each mouse.PATENT Attorney Docket No.081906-1470154-254010PC Client Ref. No. SF2023-176 Example 2 – CD27-based CAR-T Demonstrates Anti-Myeloma Activity in Cell Lines

[0136] To target CD70, 10 unique CD70-targeting CAR-T constructs were designed as discussed in Example 1 above. Nine of these constructs were scFv-based binders derived from therapeutic antibody fragments currently in preclinical or clinical development. See, e.g., International Application Publication No. WO 2021 / 095011, U.S. Patent Application Publication No. 2019 / 0233528, Chinese Patent No. 109021106, European Patent No. 2686347. Using a structure-guided design based on co-crystal structure of CD70 with its natural ligand CD27 (PDB: 7KX0), a novel truncated fragment of CD27 (SEQ ID NO:2) was designed as an alternative CD70 binding domain to the scFv-based binders.

[0137] All CD70-targeting CARs were efficacious in in vitro cytotoxicity assays targeting MM cell lines AMO1, MM1.S, LP1, and RPMI-8226 as discussed in Example 1 above. Figures 2A-C show that the CD27-based CD70-targeting CAR-T (WT CD27-CAR-2 sequence; SEQ ID NO: 17) had greater cytotoxicity than the anti-BCMA CAR-T positive control (“BCMA CAR”) and the scFv-based binder 3 CAR (“Binder 3”) in dose-response experiments with AMO1 (Figure 2A), MM.1S (Figure 2B) and RPMI-8226 (Figure 2C) cell lines. Example 3 – CD27-based CAR-T Demonstrates Anti-Lymphoma Activity in Cell Line

[0138] The CD27-based CD70-targeting CAR-T (WT CD27-CAR-2 sequence; SEQ ID NO: 17) was also tested in HuT 78 lymphoma cell line as discussed in Example 1 above. After a 24-hour incubation period and with n=3, the CD27-based CD70-targeting CAR-T (CD27 based) demonstrated 100% cytotoxicity at 1:1 ET ratio (Figure 2D). The control Empty CAR-T (Empty.EQ.28z) and anti-BCMA CAR-T negative control (BCMA-VHH.CD8.bbz) resulted in low toxicity across all effector to tumor (E:T) ratios (Figure 2D). Example 4 – A Substantial Portion The CRD-1 Region CD27 is Necessary For CAR-based Cytotoxicity

[0139] In order to determine the whether the first cysteine-rich domain (CRD-1) of CD27 (i.e., amino acids 1-43 of CD27) affected the function of a CD27-based CAR (WT CD27-CAR-2 sequence; SEQ ID NO: 17), six truncated CD27-based CARs (based on WT CD27-CAR-2 sequence; SEQ ID NO: 17) were generated. Each of these CARs had a different length of amino acids that were removed from the N-terminal end of the binding region. Most of the truncationsPATENT Attorney Docket No.081906-1470154-254010PC Client Ref. No. SF2023-176 removed portions of CRD-1, while the shortest truncation removed CRD-1 entirely and contained only CRD-2 (amino acids 44-85 of CD27) and CRD-3 (amino acids 86-101 of CD27) as binding regions for CD70. These truncated CARs are enumerated T1 to T6 and are shown in Table 1 below. The numbering scheme used is according to PDB: 7KX0. Table 1. CD27 Truncation Mutants. Mutant Truncated e tide

[0140] The cytotoxicity of T1-T6 were tested in the NOMO-1 acute myeloid leukemia cell line and the AMO1 MM cell line as discussed in Example 1 above. After 24 hours of incubation, T1- T6 had no cytotoxic effect when compared to the CD27-based CAR-T that comprised the CD27 variant SEQ ID NO: 2 (WT CD27 CAR-2 sequence SEQ ID NO: 17), even at effector:target cell ratios where the variant CD27 (WT CD27 CAR-2 sequence SEQ ID NO: 17; comprising SEQ ID NO: 2) CAR displayed nearly 100% cytotoxicity. See Figures 3A and 3B. In the NOMO-1 leukemia cell line, T1-T6 were even less toxic than the empty CAR control which did not have any extracellular domain for binding CD70. The results indicate that at least a substantial portion of the CRD-1 region of the CD27 protein is necessary for CAR-based cytotoxicity. Removal of as few as 23 amino acids from the N-terminal end of the CRD-1 region in addition to the first 22 amino acids of wildtype CD27 (SEQ ID NO: 1) (which adds up to the deletion of amino acids 1- 45 of wildtype CD27 (SEQ ID NO: 1)) leads to complete elimination of CAR activity. Example 5 – CAR-T Constructs Against Tumors in Mice

[0141] The top 3 performing scFv-based CAR-Ts – “scFv 2,” “scFv 3,” and “scFv 4” in Figure 4 – as well as the new natural ligand, CD27 CAR-T – “CD27 based” in Figure 4 – were selected for in vivo studies with mice bearing MM.1S tumors, as discussed in Example 1 above. The CD27-PATENT Attorney Docket No.081906-1470154-254010PC Client Ref. No. SF2023-176 based CAR-T (WT CD27-CAR-2 sequence; SEQ ID NO: 17) and all 3 scFv CAR-Ts prolonged survival of MM.1S-bearing mice compared to the “Empty CAR” control (n=5 mice / arm, p<0.005 by log-rank). Mice treated with the BCMA CAR-T control (“BCMA CAR”) also had prolonged survival compared to the “Empty CAR” control. Remarkably, the CD27-based CAR showed superior expansion (50-85x, p< 0.0001 by ANOVA) and persistence compared to scFv-based CAR-Ts (Figure 4). These results underscore that targeting CD70 using a natural ligand-based CAR-T represents a promising preclinical candidate for treatment of relapsed and high-risk MM. Example 6 – CD27 Mutants with Varying Affinity for CD70

[0142] CD27 mutants were designed and analyzed as discussed in Example 1 above. Two CD27 variants that were predicted to have increased affinity for CD70 were MPN 945, which had the R85L, N88E, K115S, E116L, and E119H mutations; and MPN 181, which had the Q111L and K115S mutations. Two mutants that were predicted to bind to CD70 with less affinity were N88A and the 3x Ala, which had the N88A, K115A, and E119A mutations. These CD27 mutants were introduced to the CD27-based CAR-1 sequence SEQ ID NO: 12).  Example 7 – CD27 Mutations Increase Tumor Lysis

[0143] Mutations were introduced to the CD27-based CAR (WT CD27-CAR-1 sequence; SEQ ID NO: 12) and the mutant CAR-Ts (SEQ ID NOS: 22-24) were tested for improved efficacy in tumor lysis as discussed in Example 1 above. The mutations were selected based on energetic modeling of the CD27:CD70 interface. Mutations designed to decrease affinity of CD27 for CD70, i.e., N88A (SEQ ID NO:4) and the N88A, K115A, and E119A combination (3x Ala; SEQ ID NO: 6), led to improved anti-tumor cytotoxicity compared to the CD27 wildtype fragment of amino acids 23-127 (SEQ ID NO:2). Meanwhile, mutations designed to increase affinity, i.e., the Q111L and K115S combination (MPN 181; SEQ ID NO:8), and the R85L, N88E, K115S, E116L, and E119H combination (MPN 945; SEQ ID NO:10) led to decreased cytotoxicity. Findings were reproducible across multiple-CD70+ tumor cell lines and two different T-cell donors. Results are shown in Figures 5A-D. Compared to the WT CAR-T (WT CD27-CAR-1 sequence; SEQ ID NO: 12), mutant CAR-Ts comprising a CD27 variant with decreased affinity for CD70, i.e., N88A (SEQ ID NO: 22) and 3x Ala (SEQ ID NO: 23), demonstrated greater % tumor lysis, whereas mutant CAR-Ts comprising a CD27 variant with increased affinity for CD70, i.e., MPN 945 (SEQ ID NO: 24), demonstrated lower % tumor lysis.PATENT Attorney Docket No.081906-1470154-254010PC Client Ref. No. SF2023-176 Example 8 – CD27 Mutant CAR-T Variants Reduce Tumor Burden in Mice

[0144] To assess and validate the efficacy of engineered CD27-based CAR-T cells, three CD27- based CAR-T variants (N88A, SEQ ID NO: 22; 3x Ala, SEQ ID NO: 23; and MPN 945, SEQ ID NO: 24), along with the wildtype (WT) CD27-based CAR-T cells (WT CD27-CAR-1 sequence; SEQ ID NO: 12) and a negative control of untransduced T cells, were used for in vivo studies in NSG mice bearing MOLM-14 AML cell line tumors. The CD27 mutations in the CD27-based CAR-T variants in this Example were as discussed in the preceding Examples: the N88A CAR variant (SEQ ID NO: 22; wherein the CD27 portion comprises the sequence of SEQ ID NO: 4) and the 3x Ala CAR variant (SEQ ID NO: 23; wherein the CD27 portion comprises the sequence of SEQ ID NO: 6) bind to CD70 with less affinity, while the MPN 945 CAR variant (SEQ ID NO: 24; wherein the CD27 portion comprises the sequence of SEQ ID NO: 10) binds to CD70 with greater affinity. Eight mice were tested for each type of CD27-based CAR-T cell. Tumor burden was assessed by intraperitoneal injection of D-luciferin, followed by bioluminescence imaging (BLI). BLI was performed twice weekly to assess tumor burden, and mice were sacrificed upon reaching a humane endpoint. Radiance, graphed on the Y-axis in each of Figures 6A-6E, is proportional to tumor burden in each mouse.

[0145] As shown in Figures 6A-6E, a notable reduction in tumor burden was observed in the group with WT CD27-based CAR-T cells (Figure 6B; WT CD27-CAR-1 sequence; SEQ ID NO: 12), with eventual tumor relapse observed, compared to the group with the negative control of untransduced T cells (Figure 6A). Additionally, in each of the groups with the engineered CD27- based CAR-T variants (Figures 6C-6E; N88A CD27-CAR sequence is the sequence of SEQ ID NO: 22; 3x Ala CD27-CAR sequence is the sequence of SEQ ID NO: 23; MPN 945 CD27-CAR sequence is the sequence of SEQ ID NO: 24), further reduction in tumor burden was observed when compared to the WT CD27 group (WT CD27-CAR-1 sequence; SEQ ID NO: 12), with the N88A CAR-T treatment group (SEQ ID NO: 22) displaying the greatest relative improvement, with no tumor relapse observed in the surviving mice at the end of the study period. Example 9 – Conclusion

[0146] The Examples demonstrate that targeting CD70 with CD27-based CAR-Ts is a promising cellular therapeutic strategy for MM, including having high utility in high-risk patients after BCMA CAR-T relapse. Of the CD27-based CD70-targeting CAR-Ts that were tested, thePATENT Attorney Docket No.081906-1470154-254010PC Client Ref. No. SF2023-176 variants with (1) N88A (SEQ ID NO: 22) and (2) the N88A, K115A, and E119A combination (3x Ala; SEQ ID NO: 23) demonstrated improved tumor cell killing in in vitro cytotoxicity assays (Figures 5A-5D) and in an in vivo mouse AML xenograft model (Figures 6A-6E) compared to the native CD27 sequence (SEQ ID NO: 12). The N88A CAR-T variant (SEQ ID NO: 22) also demonstrated the most significant reduction in tumor burden in an AML mouse model, with no tumor relapse observed in the surviving mice at the end of the study period. These CAR-Ts represent a new therapeutic approach for treating these forms of cancer and provide new, powerful treatment options in this immunotherapy space.   INFORMAL SEQUENCE LISTING

[0147] Sequences as identified in this disclosure are listed below. Unless noted otherwise, amino acid sequences are shown in the N-terminus to C-terminus direction and nucleic acid sequences are in the 5’ to 3’ direction. Amino acid substitutions are bolded and underlined. SEQ Description Sequence ID NO A C L Q H L S C A G G GPATENT Attorney Docket No.081906-1470154-254010PC Client Ref. No. SF2023-176 CTGAGTGTGCCTGTCGCAATGGCTGGCAGTGCAG GGACAAGGAGTGCACCGAGTGTGATCCTCTTCCA H L S C A G G G A H L S C A G G G H LPATENT Attorney Docket No.081906-1470154-254010PC Client Ref. No. SF2023-176 181; mutations are indicated with bold and C A G G G G H L S C A G G G C K L P Q KPATENT Attorney Docket No.081906-1470154-254010PC Client Ref. No. SF2023-176 Signal sequence of WT MALPVTALLLPLALLLHA CD27-CAR-1 D A A R V E K P E K H TPATENT Attorney Docket No.081906-1470154-254010PC Client Ref. No. SF2023-176 sequence of WT CD27- KPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKV CAR-2 SNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTK P E A C K L P Q KPATENT Attorney Docket No.081906-1470154-254010PC Client Ref. No. SF2023-176 3x Ala CD27-CAR MALPVTALLLPLALLLHAAPKSCPERHYWAQGKLC sequence (comprising CQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPD G K L P Q K C L P Q KPATENT Attorney Docket No.081906-1470154-254010PC Client Ref. No. SF2023-176 14 Hinge and See SEQ ID NO: 14 transmembrane

[0148] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

PATENT Attorney Docket No.081906-1470154-254010PC Client Ref. No. SF2023-176 WHAT IS CLAIMED IS:

1. A nucleic acid encoding a polypeptide comprising an extracellular target- binding domain that binds to human CD70, wherein the polypeptide comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:

2.

2. The nucleic acid of claim 1, wherein the polypeptide does not comprise wildtype full-length CD27 (SEQ ID NO: 1).

3. The nucleic acid of claim 1, wherein the amino acid sequence is at least 95% identical to SEQ ID NO: 1 and comprises at least one amino acid substitution compared to wildtype CD27.

4. The nucleic acid of any one of claims 1-3, wherein the amino acid sequence comprises an amino acid substitution at or corresponding to one or more of positions 63, 66, 89, 93, 94 and 97 of SEQ ID NO:

2.

5. The nucleic acid of claim 4, wherein the amino acid sequence comprises an amino acid substitution at or corresponding to position 66 selected from N66A, N66C, N66D, N66E, N66F, N66G, N66H, N66I, N66K, N66L, N66M, N66P, N66Q, N66R, N66S, N66T, N66V, N66W, and N66Y.

6. The nucleic acid of claim 4, wherein the amino acid sequence comprises an amino acid substitution at or corresponding to position 63 selected from R63A, R63C, R63D, R63E, R63F, R63G, R63H, R63I, R63K, R63L, R63M, R63N, R63P, R63Q, R63S, R63T, R63V, R63W, and R63Y.

7. The nucleic acid of claim 4, wherein the amino acid sequence comprises an amino acid substitution at or corresponding to position 89 selected from Q89A, Q89C, Q89D, Q89E, Q89F, Q89G, Q89H, Q89I, Q89K, Q89L, Q89M, Q89N, Q89P, Q89R, Q89S, Q89T, Q89V, Q89W, and Q89Y.

8. The nucleic acid of claim 4, wherein the amino acid sequence comprises an amino acid substitution at or corresponding to position 93 selected from K93A, K93C, K93D,PATENT Attorney Docket No.081906-1470154-254010PC Client Ref. No. SF2023-176 K93E, K93F, K93G, K93H, K93I, K93L, K93M, K93N, K93P, K93Q, K93R, K93S, K93T, K93V, K93W, and K93Y.

9. The nucleic acid of claim 4, wherein the amino acid sequence comprises an amino acid substitution at or corresponding to position 94 selected from E94A, E94C, E94D, E94F, E94G, E94H, E94I, E94K, E94L, E94M, E94N, E94P, E94Q, E94R, E94S, E94T, E94V, E94W, and E94Y.

10. The nucleic acid of claim 4, wherein the amino acid sequence comprises an amino acid substitution at or corresponding to position 97 selected from E97A, E97C, E97D, E97F, E97G, E97H, E97I, E97K, E97L, E97M, E97N, E97P, E97Q, E97R, E97S, E97T, E97V, E97W, and E97Y.

11. The nucleic acid of any one of claims 1-10, wherein the polypeptide is a chimeric antigen receptor (CAR).

12. The nucleic acid of claim 11, wherein the CAR comprises a hinge domain, a transmembrane domain, and a signaling domain.

13. The nucleic acid of claim 12, wherein the hinge domain comprises a human immunoglobulin hinge region, a hinge region from CD8 alpha, CD4, CD28, PD1, CD152, and CD7, or a mutant thereof.

14. The nucleic acid of claim 13, wherein the hinge domain is IgG4 EQ.

15. The nucleic acid of any one of claims 12-14, wherein the transmembrane domain comprises all or part of a transmembrane domain of the alpha, beta or zeta chain of the T-cell receptor, CD28, CD27, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD 11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2R beta, IL2R gamma, IL7R a, ITGAl, VLAl, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDl la, LFA-1, ITGAM, CDl lb, ITGAX, CDl lc, ITGB 1, CD29, ITGB2, CD 18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84,PATENT Attorney Docket No.081906-1470154-254010PC Client Ref. No. SF2023-176 CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100, (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME, (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKG2D, or NKG2C.

16. The nucleic acid of any one of claims 12-15, wherein the hinge and transmembrane domains comprise a combined amino acid sequence of SEQ ID NO: 14 or 20.

17. The nucleic acid of any one of claims 12-16, wherein the signaling domain comprises a CD28 co-stimulatory domain and / or a CD3 zeta activation domain.

18. The nucleic acid of any one of claims 12-16, wherein the signaling domain comprises an activation domain from CD3 zeta, common FcR gamma, Fc gamma Rlla, FcR beta (Fc Epsilon Rib), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, or DAP12.

19. The nucleic acid of any one of claims 12-18, wherein the signaling domain comprises a co-stimulatory domain from CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, or a ligand that binds to CD83.

20. The nucleic acid of any one of claims 17-19, wherein the co-stimulatory domain comprises an amino acid sequence of SEQ ID NO:

15.

21. The nucleic acid of any one of claims 17-20, wherein the co-stimulatory domain comprises an amino acid sequence of SEQ ID NO:

16.

22. The nucleic acid of any one of claims 11-21, wherein the CAR comprises an amino sequence that is at least 95% identical to the amino acid of SEQ ID NO: 12, 17, 22, 23, or 24.

23. The nucleic acid of any one of claims 11-21, wherein the CAR comprises an amino sequence of SEQ ID NO: 12, 17, 22, 23, or 24.

24. A vector comprising the nucleic acid of any one of claims 1-23.PATENT Attorney Docket No.081906-1470154-254010PC Client Ref. No. SF2023-176 25. A cell comprising the polypeptide encoded by the nucleic acid of any one of claims 1-23.

26. The cell of claim 25, wherein the cell is a human T cell, natural killer (NK) cell, macrophage, monocyte, B cell, gamma / delta T cell, natural killer T (NKT) cells induced pluripotent stem cell, hematopoietic stem cell, myeloid progenitor cell, or lymphoid progenitor cell.

27. A composition comprising (a) the cell of claim 25 or 26 and (b) a pharmaceutically acceptable carrier.

28. A method of introducing a nucleic acid encoding the polypeptide into a cell, the method comprising: providing the cell; and introducing the nucleic acid of any one of claims 1-23 or the vector of claim 24 into the cell to form a cell expressing the polypeptide.

29. The method of claim 28, wherein the cell is a human cell, optionally an immune cell, and the providing comprises obtaining the cell from a human.

30. The method of claim 28 or 29, further comprising introducing the cell expressing the polypeptide into a human.

31. The method of claim 30, wherein the cell is introduced into the human from which the cell was obtained.

32. A method of treating cancer or an autoimmune disease in a patient, the method comprising administering to the subject a pharmaceutically effective amount of the composition of claim 27.

33. The method of claim 32, wherein cells of the cancer are CD70-expressing cells.PATENT Attorney Docket No.081906-1470154-254010PC Client Ref. No. SF2023-176 34. The method of claim 32 or 33, wherein the cancer is leukemia, acute myeloid leukemia (AML), B cell acute lymphoblastic leukemia (B-ALL), B cell chronic lymphocytic leukemia (B-CLL), hairy cell leukemia, B-cell prolymphocytic leukemia, non- Hodgkin’s B cell lymphoma, diffuse large B cell lymphoma (DLBCL), follicular lymphoma, follicle center lymphoma, mantle cell lymphoma, Burkitt lymphoma (BL), Waldenstrom macroglobulinemia, Hodgkin lymphoma, multiple myeloma (MM), T cell leukemia, anaplastic large cell lymphoma, peripheral T cell lymphoma, cutaneous T cell lymphoma, extranodal natural killer (NK) / T cell lymphoma, Epstein-Barr virus associated T cell lymphoma, T-cell acute lymphoblastic leukemia, renal cell carcinoma, nasopharyngeal carcinoma, glioblastoma, melanoma, lung carcinoma, cervix carcinoma, breast carcinoma, ovarian carcinoma, mesothelioma, metastatic cancers, lung carcinoma, pancreatic carcinoma, or osteosarcoma.

35. The method of claim 32, wherein the autoimmune disease is systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), inflammatory bowel disease, multiple sclerosis, Sjoren’s syndrome, or systemic sclerosis.

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