Chimeric antigen receptor with spacer domains derived from human igg

Abstract

The present disclosure is related to chimeric antigen receptors (CARs) comprising Immunoglobulin G (IgG) derived spacers, e.g., a human IgG hinge and a human IgG Fc derived CAR spacer. IgGl derived spacers confer improved properties to the CARs, e.g., increased cytokine release with respect to CARs not comprising IgG derived spacers. Also provided are cells expressing CARs comprising IgG derived spacers regions and methods to use the CARs to treat diseases or disorders, e.g., cancer.

Description

PCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1CHIMERIC ANTIGEN RECEPTOR WITH SPACER DOMAINS DERIVED FROM HUMAN IgGCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of United States Provisional Application Serial Numbers 63 / 654,191 filed on May 31, 2024. The entire contents of this application is incorporated herein by reference in its entirety.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on March 5, 2025, is named JBI6913WOPCTl_SL.xml and is 53,811 bytes in size.TECHNICAL FIELD

[0003] The present disclosure provides chimeric antigen receptors (CAR) comprising a spacer derived from human IgG hinge and Fc regions.BACKGROUND:

[0004] Adoptive immunotherapy using chimeric antigen receptor (CAR) expressing T cells is a promising cancer treatment, because these cells can directly recognize and kill antigenexpressing tumor cells in a human leukocyte antigen (HLA)-independent manner. However, besides a careful choice of the target tumor associated antigen, this therapeutic approach is highly dependent on the optimal molecular design of the CAR.

[0005] Accordingly, there is a need for methodologies that allow the systematic optimization of the extracellular spacer in CARs to allow for maximum CAR T cell efficacy.SUMMARY

[0006] In one aspect, provided herein is a chimeric antigen receptor (CAR) comprising (i) an extracellular binding domain that binds to a tumor-associated antigen;PCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1(ii) a transmembrane domain;(iii) an intracellular domain; and,(iv) a spacer located between the extracellular binding domain and the transmembrane domain comprising an amino acid sequence derived from a hinge region and a crystallizable fragment (Fc) of a human immunoglobulin G1 isotype (IgGl) .

[0007] In some embodiments, the CAR is designed as a second-generation CAR.

[0008] In some embodiments, the transmembrane domain comprises a CD8a transmembrane region (CD8a-TM) polypeptide.

[0009] In some embodiments, the CD8a-TM polypeptide comprises an amino acid sequence of SEQ ID NO: 8.

[0010] In some embodiments, the intracellular domain comprises a TNF receptor superfamily member 9 (CD137) component and / or a T-cell surface glycoprotein CD3 zeta chain (CD3z) component.

[0011] In some embodiments, the CD 137 component comprises an amino acid sequence of SEQ ID NO: 9.

[0012] In some embodiments, the CD3z component comprises an amino acid sequence of SEQ ID NO: 10.

[0013] In some embodiments, the spacer comprises an amino acid sequence derived from the hinge, CH2, and CH3 regions of the human IgGl .

[0014] In some embodiments, the spacer comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 6.

[0015] Also provided are polynucleotides encoding the CARs described herein.

[0016] Also provided are vectors comprising the polynucleotides encoding the CARs described herein.

[0017] Also provided are cells genetically modified to express the CARs described herein.

[0018] In some embodiments, the cells that are genetically modified to express the CARs described herein are T cells.

[0019] Also provided are compositions comprising the CARs described herein.

[0020] In certain embodiments, provided is a pharmaceutical comprising the composition or the cells comprising the CARs described herein.PCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1

[0021] Also provided are methods for improving one or more properties of a CAR comprising inserting a CAR spacer between the extracellular binding domain and the transmembrane domain comprising an amino acid sequence derived from a hinge region and a crystallizable fragment (Fc) of a human immunoglobulin G1 isotype (IgGl).

[0022] In some embodiments, the spacer comprises an amino acid sequence derived from the hinge, CH2, and CH3 regions of the human IgGl .

[0023] In some embodiments, the spacer comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 6.

[0024] In some embodiments, the one or more improved properties is increased secretion of one or more cytokines or increased tumor cell conjugation.

[0025] In some embodiments, the cytokine is an interferon.

[0026] In some embodiments, the interferon is interferon-gamma.

[0027] In some embodiments, the cytokine is a tumor necrosis factor.

[0028] In some embodiments, the tumor necrosis factor is tumor necrosis factor-alpha

[0029] Also provided are methods of treating cancer in a subject in need thereof comprising administering to the subject an effective amount of the cells described herein.

[0030] Also provided are methods of preparing populations of cells for a therapy comprising transducing a population of cells isolated from a subject with the polynucleotides or the vectors described herein.

[0031] Also provided is a chimeric antigen receptor (CAR) comprising:(i) an extracellular binding domain that binds to a tumor-associated antigen;(ii) a transmembrane domain; and(iii) an intracellular domain, the improvement comprising a spacer comprising SEQ ID NO: 6.BRIEF DESCRIPTION OF THE FIGURES

[0032] FIG. 1A and FIG. IB show graphical depictions of the process of designing and testing affinity and spacer variant CARs for T cell effector function. FIG. 1 A shows that each CAR was derived from a parental monoclonal antibody later confirmed for proper folding as an scFv-Fc fusion construct. The antigen binding domain was then CDR sequence-manipulated to create aPCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1 panel of mutant scFv constructs. Each scFv was then measured for antigen binding to identify affinity variant mutants. The selected affinity variant scFvs were paired with one of three spacer designs and reformatted as CARs for functional testing. FIG. IB shows that affinity and spacer variant CAR T cells were evaluated in a series of assessments to compare CAR design influence on receptor and cellular avidity as well as T cell effector function.

[0033] FIG. 2A, FIG. 2B, FIG. 2C, and FIG 2D display the assessment of binding affinity in engineered scFv-Fc constructs targeting DLL3 and BCMA antigens. This study validates the antigen binding capability of scFv components against DLL3 and BCMA by employing DLL3+ SHP-77 tumor cells for anti-DLL3 scFv-Fc constructs and BCMA+ H929 tumor cells for anti- BCMA scFv-Fc constructs. Measurement techniques included flow cytometry and SPR for precise evaluation of antigen binding. FIG. 2A shows the flow cytometry analysis of Anti-DLL3 scFv binding to DLL3+ SHP-77 tumor cells. FIG. 2B displays SPR measurements depicting Anti-DLL3 scFv binding to recombinant human DLL3. FIG. 2C shows the flow cytometry evaluation of Anti-BCMA scFv binding to BCMA+ H929 tumor cells. FIG. 2D shows the SPR analysis showcasing Anti-BCMA scFv binding to recombinant human BCMA.

[0034] FIG. 3A, FIG.3B, FIG. 3C, and FIG 3D illustrate the engineering and evaluation of Anti- DLL3 and Anti-BCMA affinity variants with various spacers. Lentiviral transduction was employed to introduce designed anti-DLL3 and anti-BCMA chimeric antigen receptor (CAR) constructs with three distinct spacers. Surface CAR expression post-transduction was analyzed using flow cytometry. FIG. 3A shows the anti-DLL3 CAR design, featuring the pairing of four affinity variant single-chain variable fragments (scFvs) with CD8 (short), CH2-CH3 (intermediate), or CH2-CH3-CH2-CH3 (long) spacer domains, coupled with CD8 transmembrane (TM), 41BB costimulatory, and CD3(^ signaling domains. FIG 3B shows the anti-BCMA CAR design, showcasing the pairing of three affinity variant scFvs with CD8 (short), CH2-CH3 (intermediate), or CH2-CH3-CH2-CH3 (long) spacer domains, coupled with CD8 transmembrane, 4-1BB costimulatory, and CD3(^ signaling domains. In FIG. 3C, the anti- DLL3 CAR surface expression is shown represented as %CAR+ T cells, and surface density measured by flow cytometry. In FIG. 3D, the anti-BCMA CAR surface expression is represented as %CAR+ T cells, and surface density measured by flow cytometry. In all, the observations from FIG. 3A, FIG.3B, FIG. 3C, and FIG 3D suggest that high-affinity CAR designs for DLL3 and BCMA CARs do not exhibit a discernible decrease in CAR expression."PCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1

[0035] FIG. 4A, FIG.4B, FIG. 4C, FIG 4D, FIG. 4E, and FIG 4F show the exploration of anti- DLL3 and Anti-BCMA affinity variants with three spacers, in particular, the evaluation of CAR clustering and tumor cell conjugation. FIG. 4A depicts the clustering of anti-DLL3 CARs, measured by binding to soluble recombinant human DLL3 complexes, reveals affinity-dependent advantages in clustering, while spacer design shows no significant impact. FIG. 4B depicts the clustering of anti-BCMA CARs, measured by binding to soluble recombinant human BCMA complexes, demonstrates affinity-dependent advantages in clustering, with no notable effect from spacer design. FIG. 4C and FIG. 4D show that the tumor cell conjugation assays for anti- DLL3 and anti-BCMA CARs, respectively, indicate enhanced cellular avidity for CARs paired with CH2-CH3 (intermediate) spacers compared to those paired with CD8 (short) or tandem CH2-CH3 (long) spacers. FIG 4E and FIG. 4F show that CAR signaling measurements for anti- DLL3 and anti-BCMA CARs unveil the impact of both affinity and spacer design. CAR signaling is defined as % dual expression of activation markers, Nur77 (signal 1), and NF-KB (signal 2). In all, these findings suggest that CAR signaling diminishes when CAR affinity is weaker than lOnM KD. Responses to tumor cells are most pronounced for short spacer CARs and decrease as spacer length increases. As expected, spacer differences are less profound against antigen complexes due to the absence of a mature immune synapse. These results underscore the influence of spacer length on CAR signaling, with variations observed only in scenarios where synaptic length is altered.

[0036] FIG.5A and FIG. 5B illustrate the functional evaluation of high-affinity DLL3 CARs with optimal spacer against low-affinity DLL3 CAR. Tumor targets were co-cultured with CAR- T cells to assess the cytotoxicity and polyfunctional response of anti-DLL3 CARs against tumor spheroids. Testing involved various effector-to-target (E: T) ratios, measuring tumor killing, and quantifying IFN-g, IL-2, and TNF-a. In FIG. 5A, the cytotoxicity and CAR T cell proliferation of DLL3 CAR designs against tumor targets were examined using both high and low E: T ratios. FIG. 5B shows the polyfunctional cytokine production by CAR T cells after co-culture with tumor targets was also measured. DLL3 CARs paired with intermediate-length (IgGl CH2-CH3) spacers exhibited equivalent or superior tumor lysis and T cell expansion compared to those with short or long spacers. Additionally, low-affinity DLL3-CAR demonstrated lower cytotoxicity compared to high-affinity CAR. These findings suggest that pairing high-affinity CARs withPCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1 intermediate spacers results in maximal cytotoxicity and increased production of IFN-g and TNF-a compared to low-affinity CARs.

[0037] FIG.6A and FIG 6B shows the functional assessment of high-affinity BCMA CARs with optimal spacer against low-affinity BCMA CAR. Tumor targets were co-cultured with CAR-T cells to examine the cytotoxicity and polyfunctional response of anti-BCMA CARs against tumor spheroids. Testing involved low and high effector-to-target (E: T) ratios, with measurements taken for tumor killing, IFN-g, IL-2, and TNF-a. In FIG. 6A, the cytotoxicity and CAR T cell proliferation of BCMA CAR designs against tumor targets were analyzed at both high and low E: T ratios. FIG. 6B shows that Polyfunctional cytokine production by CAR T cells after co-culture with tumor targets was also assessed. BCMA CARs paired with intermediatelength (IgGl CH2-CH3) spacers demonstrated equivalent or superior tumor lysis and T cell expansion compared to those with short or long spacers. Notably, minimal functional differences were observed for the affinity variant CARs, indicating reduced function only when CAR affinity is weaker than lOOnM (KD). The results indicate that high-affinity CARs paired with intermediate spacers yielded comparable cytotoxicity and IFN-g and TNF-a production to low- affinity CARs, affirming the functional efficacy of high-affinity CARs.DETAILED DESCRIPTION

[0038] The present disclosure is directed to CARs comprising spacers derived from amino acid sequences of the hinge region of a human immunoglobulin G1 isotype (IgGl) and the crystallizable fragment (Fc) of a human IgGl, polynucleotides encoding such CARs, cells expressing the CARs, and methods for their use. In some aspects, the CAR spacer comprises a subsequence from human IgGl hinge and human IgGl Fc regions, e g., a CAR spacer can comprise a hinge and CH2 and / or CH3 subsequences.

[0039] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to the particular compositions or process steps described, as such can, of course, vary. As will be apparent to those of skill in the art upon reading this disclosure, each of the individual aspects described and illustrated herein has discrete components and features which can be readily separated from or combined with the features of any of the other severalPCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1 aspects without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0040] Headings provided herein are not limitations of the various aspects of the disclosure, which can be defined by reference to the specification as a whole. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0041] Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0042] DEFINITIONS

[0043] In order that the present description can be more readily understood, certain terms are first defined. Except as otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout the detailed description.

[0044] It is to be noted that the term "a" or "an" entity refers to one or more of that entity; for example, "an amino acid sequence," is understood to represent one or more amino acid sequences. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein. It is further noted that the claims can be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a negative limitation.

[0045] Furthermore, "and / or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or" as used in a phrase such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).PCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1

[0046] It is understood that wherever aspects are described herein with the language "comprising," otherwise analogous aspects described in terms of "consisting of and / or "consisting essentially of are also provided.

[0047] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei- Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.

[0048] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Where a range of values is recited, it is to be understood that each intervening integer value, and each fraction thereof, between the recited upper and lower limits of that range is also specifically disclosed, along with each subrange between such values. The upper and lower limits of any range can independently be included in or excluded from the range, and each range where either, neither or both limits are included is also encompassed within the disclosure. Thus, ranges recited herein are understood to be shorthand for all of the values within the range, inclusive of the recited endpoints. For example, a range of 1 to 10 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0049] Where a value is explicitly recited, it is to be understood that values which are about the same quantity or amount as the recited value are also within the scope of the disclosure. Where a combination is disclosed, each subcombination of the elements of that combination is also specifically disclosed and is within the scope of the disclosure. Conversely, where different elements or groups of elements are individually disclosed, combinations thereof are also disclosed. Where any element of a disclosure is disclosed as having a plurality of alternatives, examples of that disclosure in which each alternative is excluded singly or in any combination with the other alternatives are also hereby disclosed; more than one element of a disclosure can have such exclusions, and all combinations of elements having such exclusions are hereby disclosed.PCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1

[0050] Nucleotides are referred to by their commonly accepted single-letter codes. Unless otherwise indicated, nucleotide sequences are written left to right in 5' to 3' orientation. Nucleotides are referred to herein by their commonly known one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Accordingly, ‘a’ represents adenine, ‘c’ represents cytosine, ‘g’ represents guanine, ‘t’ represents thymine, and ‘u’ represents uracil.

[0051] Amino acid sequences are written left to right in amino to carboxy orientation. Amino acids are referred to herein by either their commonly known three letter symbols or by the one- letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0052] The term "about" is used herein to mean approximately, roughly, around, or in the regions of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" can modify a numerical value above and below the stated value by a variance of, e.g., 10 percent, up or down (higher or lower).

[0053] The terms "administration," "administering," and grammatical variants thereof refer to introducing a composition of the present disclosure (e.g, a polynucleotide encoding a CAR or a cell expressing a CAR), into a subject via a pharmaceutically acceptable route. The introduction of a composition of the present disclosure (e.g, a polynucleotide encoding a CAR or a cell expressing a CAR), into a subject is by any suitable route, including intratumorally, orally, pulmonarily, intranasally, parenterally (intravenously, intra-arterially, intramuscularly, intraperitoneally, or subcutaneously), rectally, intralymphatically, intrathecally, periocularly or topically.

[0054] Administration includes self-administration and the administration by another. A suitable route of administration allows the composition or the agent to perform its intended function. For example, if a suitable route is intravenous, the composition is administered by introducing the composition or agent into a vein of the subject.

[0055] The term "amino acid substitution" refers to replacing an amino acid residue present in a parent or reference sequence (e.g, a wildtype sequence) with another amino acid residue. An amino acid can be substituted in a parent or reference sequence (e.g, a wild-type polypeptide sequence), for example, via chemical peptide synthesis or through recombinant methods known in the art. A reference to a "substitution at position X" refers to the substitution of an amino acidPCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1 present at position X with an alternative amino acid residue. Substitution patterns can be described according to the schema AnY, wherein A is the single letter code corresponding to the amino acid naturally or originally present at position n, and Y is the substituting amino acid residue. In other aspects, substitution patterns can be described according to the schema An(YZ), wherein A is the single letter code corresponding to the amino acid residue substituting the amino acid naturally or originally present at position n, and Y and Z are alternative substituting amino acid residues that can replace A.

[0056] As used herein, the term "approximately," as applied to one or more values of interest, refers to a value that is similar to a stated reference value. The term "approximately" refers to a range of values that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0057] As used herein, the term "conserved" refers to nucleotides or amino acid residues of a polynucleotide sequence or polypeptide sequence, respectively, that are those that occur unaltered in the same position of two or more sequences being compared. Nucleotides or amino acids that are relatively conserved are those that are conserved amongst more related sequences than nucleotides or amino acids appearing elsewhere in the sequences.

[0058] In some aspects, two or more sequences are said to be "completely conserved" or "identical" if they are 100% identical to one another. In some aspects, two or more sequences are said to be "highly conserved" if they are at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, or at least about 95% identical to one another. In some aspects, two or more sequences are said to be "highly conserved" if they are about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, about 95% identical, about 98% identical, or about 99% identical to one another. In some aspects, two or more sequences are said to be "conserved" if they are at least about 30% identical, at least about 35% identical, at least about 40% identical, at least about 45% identical, at least about 50% identical, at least about 55%, at least about 60% identical, at least about 65% identical, at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, or at least about 95% identical to one another. In some aspects, two or more sequences are said to bePCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1"conserved" if they are about 30% identical, about 35% identical, about 40% identical, about 45% identical, about 50% identical, about 55% identical, about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, about 95% identical, about 98% identical, or about 99% identical to one another. Conservation of sequence may apply to the entire length of a polynucleotide or polypeptide or may apply to a portion, region or feature thereof.

[0059] "Derived from" as that term is used herein, indicates a relationship (e.g ., structural similarity) between a first and a second molecule. For example, in the case of a CAR spacer of the present disclosure comprising an amino acid sequence derived from a human IgGl sequence (e.g., a hinge region and an Fc domain), the sequence that is derived from the human IgGl sequence (e.g., a hinge region and an Fc domain) can comprise or consist of a full hinge, a hinge fragment, a full hinge or a fragment of an hinge plus additional residues adjacent to the hinge in a wild type IgGl (e.g., one or more amino acids from an Fc domain such as a CH2 or CH3 domain), or can comprise or consist of the full CH2 and CH3 domains, a CH2 and CH3 fragment, or a CH2 and CH3 fragment plus additional residues adjacent in a wild type IgGl (e.g., one or more amino acids from a secondary structure element, e.g., a beta-sheet, adjacent to a loop region in a CH2 or CH3 domain).

[0060] CAR spacers derived from a human IgGl (e.g., a hinge region and an Fc domain), disclosed herein also encompass sequences generated by covalently linking via peptidic bonds a hinge region derived sequence as described above, i.e., the spacer can be a polymer comprising multiple repeats of a full hinge, fragments thereof, or combinations thereof.

[0061] As used herein, the terms "extracellular binding domain" and "antibody" encompass an immunoglobulin whether natural or partly or wholly synthetically produced, and antigen-binding portions thereof. The term also covers any protein having a binding domain that is homologous to an immunoglobulin binding domain, "extracellular binding domain" and "antibody" further include a polypeptide comprising a framework region from an immunoglobulin gene or portions thereof that specifically binds and recognizes an antigen, and comprises at least one CDR. Use of the terms "extracellular binding domain" and "antibody" is meant to include whole antibodies, polyclonal, monoclonal and recombinant antibodies, portions thereof, and further includes single-chain antibodies, humanized antibodies, murine antibodies, chimeric, mouse-human, mouse-primate, primate-human monoclonal antibodies, anti-idiotype antibodies, antibodyPCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1 constructs, such as, e.g ., scFv, (SCFV)2, Fab, Fab', and F(ab')2, F(abl)2, Fv, dAb, and Fd, disulfide-linked Fvs (dsFcs), and antibody-related polypeptides.

[0062] In some aspects, an "extracellular binding portion" refers to a polypeptide sequence that makes contacts with the antigen, including but not limited to CDRs derived from an antibody.

[0063] An extracellular binding portion can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g, Hollinger and Hudson, Nature Biotechnology 23 : 1126-1136, 2005). Antigen binding portions can also be grafted into scaffolds based on polypeptides such as a fibronectin type III (Fn3)(see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide minibodies). Thus, the terms "antigen-binding domain" and "antibody" include also antibody mimics based on the scaffold of the fibronectin type III domain (monobodies), other scaffolding systems (e.g, tenascin) in which one or more CDRs are grafted, aptamers, etc.

[0180] The terms "antigenbinding domain" and "antibody" also include other suitable antigen-binding domains that can be used according to the present disclosure, e.g., nanobody, VHH antibody, DARPin (designed ankyrin repeat proteins), affibody, monobody, adnectin, alphabody, Albumin-binding domain, Adhiron, Affilin and other gamma-B crystallin-derived artificial proteins, Affimer, Affitin ( NANOFITIN™), Anticalin, Armadillo repeat proteins (ARM-repeat protein such as, e.g., b- catenin, a-importing, plakoglobin, adenomatous polyposis cob, ARMC4, ARMCX3, etc.), Atrimer (e.g, tetranectin and derived proteins), Avimer / Maxibody, Centyrin, Fynomer and other Fyn SH3 domain-derived proteins, Kunitz domain, Obody / OB-fold, Pronectin, Repebody, or any synthetic and / or computationally designed binding-protein or scaffold.

[0064] The modular architecture of antibodies has been exploited to create more than 60 different bispecific or multispecific antibody formats. Accordingly, in some aspects, the antibody can be in a format selected, e.g, from crossMab, DAF (Dual Action Fab) (two-in-one), DAF (four- in-one), DutaMab, DT-IgG, Knobs-in-holes common LC, Knobs-in-holes assembly, Charge pair, Fab-arm exchange, SEEDbody, Triomab, LUZ-Y (bispecific antibody with a leucize zipper inducing heterodimerization of two HCs), Fcab, Kl-body, Orthogonal Fab, DVD- IgG (dual variable domain IgG), IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG- 2scFv, scFv4-Ig, Zybody, DVI-IgG (four-in-one), Nanobody, Nanobody-HSA, BiTE (bispecific T cell engager), Diabody, DART (dual-affmity-retargeting), TandAb (tandem antibody),PCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1 scDiabody, scDiabody- CH3, Triple Body, Miniantibody, Minibody, TriBi minibody, scFv-CFB KIH, Fab-scFv, scFv-CH- CL-scFv, F(ab')2, F(ab')2-ScFv2, scFv-KIH, Fab-scFv-Fc, Tetravalent HC Ab, scDiabody-Fc, Diabody-Fc, Tandem scFv-Fc, Intrabody, Dock and Loeck, ImmTAC, HSAbody, scDiabody- HSA, Tandem scFv-Toxin, IgG-IgG, Cov-X-Body, and scFvl-PEG- scFv2.

[0065] "Extracellular binding domain" and "antibody" also include bispecific and multispecific antibodies so long as they exhibit the desired biological activity or function. In some aspects, the CAR of the present disclosure comprises an extracellular binding domain, e.g, an scFv.

[0066] The term "scFv" refers to a fusion protein comprising at least one antibody portion comprising a variable region of a light chain and at least one antibody portion comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked, e.g, via a synthetic linker, e.g, a short flexible polypeptide linker, and capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified, as used herein an scFv may have the VL and VH variable regions in either order, e.g., with respect to the N- terminal and C-terminal ends of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker-VL.

[0067] The term "complementarity determining region" or "CDR," as used herein, refers to the sequences of amino acids within antibody variable regions which confer antigen specificity and binding affinity. For example, in general, there are three CDRs in each heavy chain variable region (e.g, HCDR1, HCDR2, and HCDR3) and three CDRs in each light chain variable region (LCDR1, LCDR2, and LCDR3). The precise amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known schemes, including those described by Rabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. ("Kabat" numbering scheme), Al-Lazikani et al., (1997) JMB 273,927-948 ("Chothia" numbering scheme), or a combination thereof. Under the Rabat numbering scheme, in some embodiments, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Under the Chothia numbering scheme, in some embodiments, the CDR amino acids in the VH are numbered 26-32 (HCDR1),PCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT152-56 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in the VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). In a combined Rabat and Chothia numbering scheme, in some embodiments, the CDRs correspond to the amino acid residues that are part of a Rabat CDR, a Chothia CDR, or both. For instance, in some embodiments, the CDRs correspond to amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in a VH, e.g, a mammalian VH, e.g., a human VH; and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in a VL, e.g, a mammalian VL, e.g, a human VL.

[0068] The term "antigen" refers to a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically- competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA.

[0069] The term "autologous" refers to any material derived from the same individual to whom it is later to be re-introduced into the individual.

[0070] The term "Chimeric Antigen Receptor" or alternatively a "CAR" refers to a set of polypeptides, typically two in the simplest form, which when in an immune effector cell, provides the cell with specificity for a target cell, typically a cancer cell, and with intracellular signal generation. In some aspects, a CAR comprises at least an extracellular binding domain, a transmembrane domain and a cytoplasmic signaling domain (also referred to as "an intracellular domain") comprising a functional signaling domain derived from a stimulatory molecule and / or costimulatory molecule as defined below. In some aspects, the set of polypeptides are in the same polypeptide chain, e.g., comprise a chimeric fusion protein. In some aspects, the set of polypeptides are not contiguous with each other, e.g, are in different polypeptide chains. In some aspects, the set of polypeptides include a dimerization switch that, upon the presence of a dimerization molecule, can couple the polypeptides to one another, e.g, can couple an antigen binding domain to an intracellular signaling domain. In some aspects, the stimulatory molecule of the CAR is the zeta chain associated with the T cell receptor complex (CD3 zeta). In some aspects, the intracellular domain comprises a primary signaling domain (e.g, a primary signaling domain of CD3 zeta). In some aspects, the intracellular domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule defined below. InPCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1 some aspects, the costimulatory molecule is chosen from the costimulatory molecules described herein, e.g, 4-1BB, CD27, and / or CD28.

[0071] In some aspects, the CAR comprises a chimeric fusion protein comprising an extracellular binding domain, a transmembrane domain and an intracellular domain comprising a functional signaling domain derived from a stimulatory molecule, wherein the antigen-binding domain and the transmembrane domain are linked by a CAR spacer. In some aspects, the CAR comprises a chimeric fusion protein comprising an antigen-binding domain linked to a transmembrane domain via a CAR spacer and an intracellular domain comprising a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In some aspects, the CAR comprises a chimeric fusion protein comprising an extracellular binding domain linked to a transmembrane domain via a Car spacer and an intracellular domain comprising two functional signaling domains derived from one or more costimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule. In some aspects, the CAR comprises a chimeric fusion protein comprising an extracellular binding domain linked to a transmembrane domain via a CAR spacer and an intracellular domain comprising at least two functional signaling domains derived from one or more costimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule. In some aspects, the CAR comprises an optional leader sequence at the aminoterminus (N-terminus) of the CAR. In some aspects, the CAR further comprises a leader sequence at the N-terminus of the antigen-binding domain, wherein the leader sequence is optionally cleaved from the antigen-binding domain (e.g., a scFv) during cellular processing and localization of the CAR to the cellular membrane.

[0072] The term "cancer" refers to a disease characterized by the uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers are described herein and include but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer and the like. The terms "tumor" and "cancer" are used interchangeably herein, e.g., both terms encompass solid and liquid, e.g, diffuse or circulating, tumors. As used herein, the term "cancer" or "tumor" includes premalignant and malignant cancers and tumors.PCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1

[0073] The terms "tumor-associated antigen" or "tumor antigen" or variants thereof interchangeably refer to a molecule (typically protein, carbohydrate or lipid) that is preferentially expressed on the surface of a cancer cell, either entirely or as a fragment (e.g, MHC / peptide), in comparison to a normal cell, and which is useful for the preferential targeting of a pharmacological agent to the cancer cell. In some aspects, a tumor antigen is a marker expressed by both normal cells and cancer cells, e.g, a lineage marker, e.g, CD 19 on B cells. In certain aspects, the tumor antigen is derived from, cancers including but not limited to primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, leukemias, uterine cancer, cervical cancer, bladder cancer, kidney cancer and adenocarcinomas such as breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, and the like.

[0074] In some aspects, the tumor antigen is an antigen that is common to a specific proliferative disorder. In some aspects, a cancer-associated antigen is a cell surface molecule that is overexpressed in a cancer cell in comparison to a normal cell, for instance, 1-fold over expression, 2-fold overexpression, 3-fold overexpression or more in comparison to a normal cell. In some aspects, a cancer-associated antigen is a cell surface molecule that is inappropriately synthesized in the cancer cell, for instance, a molecule that contains deletions, additions or mutations in comparison to the molecule expressed on a normal cell. In some aspects, a cancer- associated antigen will be expressed exclusively on the cell surface of a cancer cell, entirely or as a fragment ( e.g ., MHC / peptide), and not synthesized or expressed on the surface of a normal cell.

[0075] A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g, aspartic acid, glutamic acid), uncharged polar side chains (e.g, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g, threonine, valine, isoleucine) and aromatic side chains (e.g, tyrosine, phenylalanine, tryptophan, histidine). Thus, if an amino acid in a polypeptide is replaced with another amino acid from the same side chain family, the substitution is considered to be conservative. In another aspect, a string of amino acids can be conservatively replaced withPCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1 a structurally similar string that differs in order and / or composition of side chain family members.

[0196] Non-conservative amino acid substitutions include those in which (i) a residue having an electropositive side chain (e.g, Arg, His or Lys) is substituted for, or by, an electronegative residue (e.g, Glu or Asp), (ii) a hydrophilic residue (e.g, Ser or Thr) is substituted for, or by, a hydrophobic residue (e.g, Ala, Leu, lie, Phe or Vai), (iii) a cysteine or proline is substituted for, or by, any other residue, or (iv) a residue having a bulky hydrophobic or aromatic side chain ( e.g ., Vai, His, lie or Trp) is substituted for, or by, one having a smaller side chain (e.g., Ala or Ser) or no side chain (e.g, Gly).

[0076] Other amino acid substitutions can also be used. For example, for the amino acid alanine, a substitution can be taken from any one of D-alanine, glycine, beta-alanine, L-cysteine and D- cysteine. For lysine, a replacement can be any one of D-lysine, arginine, D-arginine, homo arginine, methionine, D-methionine, ornithine, or D- ornithine. Generally, substitutions in functionally important regions that can be expected to induce changes in the properties of isolated polypeptides are those in which (i) a polar residue, e.g, serine or threonine, is substituted for (or by) a hydrophobic residue, e.g, leucine, isoleucine, phenylalanine, or alanine; (ii) a cysteine residue is substituted for (or by) any other residue; (iii) a residue having an electropositive side chain, e.g, lysine, arginine or histidine, is substituted for (or by) a residue having an electronegative side chain, e.g, glutamic acid or aspartic acid; or (iv) a residue having a bulky side chain, e.g, phenylalanine, is substituted for (or by) one not having such a side chain, e.g, glycine. The likelihood that one of the foregoing non-conservative substitutions can alter functional properties of the protein is also correlated to the position of the substitution with respect to functionally important regions of the protein: some non-conservative substitutions can accordingly have little or no effect on biological properties.

[0077] In the content of the present disclosure, the terms "mutation" and "amino acid substitution" as defined above (sometimes referred simply as a "substitution") are considered interchangeable.

[0078] In the context of the present disclosure, substitutions (even when they are referred to as amino acid substitution) are conducted at the nucleic acid level, i.e., substituting an amino acid residue with an alternative amino acid residue is conducted by substituting the codon encoding the first amino acid with a codon encoding the second amino acid.PCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1

[0079] As used herein, the term "homology" refers to the overall relatedness between polymeric molecules, e.g. between nucleic acid molecules (e.g. DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Generally, the term "homology" implies an evolutionary relationship between two molecules. Thus, two molecules that are homologous will have a common evolutionary ancestor. In the context of the present disclosure, the term homology encompasses both to identity and similarity.

[0080] In some aspects, polymeric molecules are considered to be "homologous" to one another if at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% of the monomers in the molecule are identical (exactly the same monomer) or are similar (conservative substitutions). The term "homologous" necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences).

[0081] As used herein, the term "identity" refers to the overall monomer conservation between polymeric molecules, e.g ., between polypeptide molecules or polynucleotide molecules (e.g. DNA molecules and / or RNA molecules). The term "identical" without any additional qualifiers, e.g, protein A is identical to protein B, implies the sequences are 100% identical (100% sequence identity). Describing two sequences as, e.g, "70% identical," is equivalent to describing them as having, e.g, "70% sequence identity."

[0082] Calculation of the percent identity of two polypeptide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g, gaps can be introduced in one or both of a first and a second polypeptide sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain aspects, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence. The amino acids at corresponding amino acid positions are then compared.

[0083] When a position in the first sequence is occupied by the same amino acid as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap,PCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1 which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.

[0084] Suitable software programs are available from various sources, and for alignment of both protein and nucleotide sequences. One suitable program to determine percent sequence identity is bl2seq, part of the BLAST suite of program available from the U.S. government's National Center for Biotechnology Information BLAST web site (blast.ncbi.nlm.nih.gov). B12seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, e.g, Needle, Stretcher, Water, or Matcher, part of the EMBOSS suite of bioinformatics programs and also available from the European Bioinformatics Institute (EBI) at www.ebi.ac.uk / Tools / psa.

[0206] Sequence alignments can be conducted using methods known in the art such as MAFFT, Clustal (ClustalW, Clustal X or Clustal Omega), MUSCLE, etc.

[0085] Different regions within a single polynucleotide or polypeptide target sequence that aligns with a polynucleotide or polypeptide reference sequence can each have their own percent sequence identity. It is noted that the percent sequence identity value is rounded to the nearest tenth. For example, 80.11, 80.12, 80.13, and 80.14 are rounded down to 80.1, while 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to 80.2. It also is noted that the length value will always be an integer.

[0086] In certain aspects, the percentage identity (%ID) or of a first amino acid sequence (or nucleic acid sequence) to a second amino acid sequence (or nucleic acid sequence) is calculated as %ID = 100 x (Y / Z), where Y is the number of amino acid residues (or nucleobases) scored as identical matches in the alignment of the first and second sequences (as aligned by visual inspection or a particular sequence alignment program) and Z is the total number of residues in the second sequence. If the length of a first sequence is longer than the second sequence, the percent identity of the first sequence to the second sequence will be higher than the percent identity of the second sequence to the first sequence.

[0087] One skilled in the art will appreciate that the generation of a sequence alignment for the calculation of a percent sequence identity is not limited to binary sequence-sequence comparisons exclusively driven by primary sequence data. It will also be appreciated thatPCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1 sequence alignments can be generated by integrating sequence data with data from heterogeneous sources such as structural data (e.g., crystallographic protein structures), functional data (e.g, location of mutations), or phylogenetic data. A suitable program that integrates heterogeneous data to generate a multiple sequence alignment is T-Coffee, available at www.tcoffee.org, and alternatively available, e.g, from the EBI. It will also be appreciated that the final alignment used to calculate percent sequence identity can be curated either automatically or manually.

[0088] As used herein, the term "similarity" refers to the overall relatedness between polymeric molecules, e.g. between polynucleotide molecules (e.g. DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Calculation of percent similarity of polymeric molecules to one another can be performed in the same manner as a calculation of percent identity, except that calculation of percent similarity takes into account conservative substitutions as is understood in the art. It is understood that percentage of similarity is contingent on the comparison scale used, i.e., whether the amino acids are compared, e.g, according to their evolutionary proximity, charge, volume, flexibility, polarity, hydrophobicity, aromaticity, isoelectric point, antigenicity, or combinations thereof.

[0089] As used herein, the terms "isolated," "purified," "extracted," and grammatical variants thereof are used interchangeably and refer to the state of a preparation of desired composition of the present disclosure, e.g., a CAR of the present disclosure, that has undergone one or more processes of purification. In some aspects, isolating or purifying as used herein is the process of removing, partially removing (e.g, a fraction) of a composition of the present disclosure, e.g, a CAR of the present disclosure from a sample containing contaminants.

[0090] In some aspects, an isolated composition has no detectable undesired activity or, alternatively, the level or amount of the undesired activity is at or below an acceptable level or amount. In other aspects, an isolated composition has an amount and / or concentration of desired composition of the present disclosure, at or above an acceptable amount and / or concentration and / or activity. In other aspects, the isolated composition is enriched as compared to the starting material from which the composition is obtained. This enrichment can be by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, atPCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1 least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.9%, at least about 99.99%, at least about 99.999%, at least about 99.9999%, or greater than 99.9999% as compared to the starting material.

[0091] In some aspects, isolated preparations are substantially free of residual biological products. In some aspects, the isolated preparations are 100% free, at least about 99% free, at least about 98% free, at least about 97% free, at least about 96% free, at least about 95% free, at least about 94% free, at least about 93% free, at least about 92% free, at least about 91% free, or at least about 90% free of any contaminating biological matter. Residual biological products can include abiotic materials (including chemicals) or unwanted nucleic acids, proteins, lipids, or metabolites.

[0092] "Nucleic acid," "nucleic acid molecule," "nucleotide sequence," "polynucleotide," and grammatical variants thereof are used interchangeably and refer to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNA molecules") or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecules"), or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix. Single stranded nucleic acid sequences refer to single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA). Double stranded DNA- DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic acid molecule, and in particular DNA or RNA molecule, refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double- stranded DNA found, inter alia , in linear or circular DNA molecules (e.g, restriction fragments), plasmids, supercoiled DNA and chromosomes. In discussing the structure of particular double- stranded DNA molecules, sequences can be described herein according to the normal convention of giving only the sequence in the 5’ to 3’ direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).

[0093] A "recombinant DNA molecule" is a DNA molecule that has undergone a molecular biological manipulation. DNA includes, but is not limited to, cDNA, genomic DNA, plasmid DNA, synthetic DNA, and semi-synthetic DNA. A "nucleic acid composition" of the disclosure comprises one or more nucleic acids as described herein.PCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1

[0094] The term "polynucleotide" as used herein refers to polymers of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof. This term refers to the primary structure of the molecule. Thus, the term includes triple-, double- and single-stranded deoxyribonucleic acid ("DNA"), as well as triple-, double- and single-stranded ribonucleic acid ("RNA"). It also includes modified, for example by alkylation, and / or by capping, and unmodified forms of the polynucleotide. More particularly, the term "polynucleotide" includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose) and polyribonucleotides (containing D- ribose), including mRNA, whether spliced or unspliced, any other type of polynucleotide which is an N- or C-glycoside of a purine or pyrimidine base, and other polymers containing normucleotidic backbones, for example, polyamide (e.g, peptide nucleic acids "PNAs") and polymorpholino polymers, and other synthetic sequence-specific nucleic acid polymers providing that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA.

[0095] In some aspects, a polynucleotide disclosed herein comprises a DNA, e.g, a DNA inserted in a vector. In other aspects, a polynucleotide disclosed herein comprises an mRNA. In some aspects, the mRNA is a synthetic mRNA. In some aspects, the synthetic mRNA comprises at least one unnatural nucleobase. In some aspects, all nucleobases of a certain class have been replaced with unnatural nucleobases (e.g, all uridines in a polynucleotide disclosed herein can be replaced with an unnatural nucleobase, e.g, 5-methoxyuridine).

[0096] The term "encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene, cDNA, or RNA, encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0097] Unless otherwise specified, a nucleotide sequence "encoding" an amino acid sequence," e.g., a polynucleotide "encoding" a CAR of the present disclosure, includes all nucleotidePCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1 sequences that are degenerate versions of each other and that encode the same amino acid sequence.

[0098] The term "expression" refers to the transcription and / or translation of a particular nucleotide sequence driven by a promoter.

[0099] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer can comprise modified amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids such as homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and creatine), as well as other modifications known in the art.

[0100] The term "polypeptide," as used herein, refers to proteins, polypeptides, and peptides of any size, structure, or function. Polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing. A polypeptide can be a single polypeptide or can be a multi-molecular complex such as a dimer, trimer or tetramer. They can also comprise single chain or multichain polypeptides. Most commonly disulfide linkages are found in multichain polypeptides. The term polypeptide can also apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid. In some aspects, a "peptide" can be less than or equal to 50 amino acids long, e.g, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

[0101] A "recombinant" polypeptide or protein refers to a polypeptide or protein produced via recombinant DNA technology. Recombinantly produced polypeptides and proteins expressed in engineered host cells are considered isolated for the purpose of the disclosure, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique. The polypeptides disclosed herein can be recombinantly produced using methods known in the art. In some aspects, the CARs of the present disclosure are recombinantly produced. In some aspects, the CARs of the present disclosure are producedPCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1 by cells, e.g., T cells, following transfection with at least one polynucleotide vector encoding a CAR of the present disclosure.

[0102] As used herein, the term "fragment" of a polypeptide (e.g, a human IgGl hinge and a human IgGl Fc.) refers to an amino acid sequence of a polypeptide that is shorter than the naturally-occurring sequence, N- and / or C-terminally deleted or any part of the polypeptide deleted in comparison to the naturally occurring polypeptide. Thus, a fragment does not necessarily need to have only N- and / or C- terminal amino acids deleted. A polypeptide in which internal amino acids have been deleted with respect to the naturally occurring sequence is also considered a fragment.

[0103] As used herein, the term "functional fragment" refers to a polypeptide fragment that retains polypeptide function. Accordingly, in some aspects, a functional fragment of an Ig hinge, retains the ability to position an antigen-binding domain (e.g, an scFv) in a CAR at a distance from a target epitope (e.g, a tumor antigen) such that the antigen-binding domain (e.g, an scFv) can effectively interact with the target epitope (e.g, a tumor antigen).

[0104] Whether a IgGl hinge fragment is a functional fragment can be assessed by any art known methods to determine the binding of the CAR comprising the spacer to a target antigen, and T-cell activation, including, e.g, Western Blots, FACS analysis, cytokine secretion analyses, cell survival analyses, etc. In certain aspects, an IgGl hinge functional fragment is a fragment that when used as a spacer in a CAR, results in a CAR with, e.g, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or about 100% of the activity of a reference CAR. As used herein, the term "reference CAR" refers to a corresponding CAR comprising no spacer.

[0105] Using known methods of protein engineering and recombinant DNA technology, variants can be generated to improve or alter the characteristics of the polypeptides. For instance, one or more amino acids can be deleted from the N-terminus or C-terminus of the secreted protein without substantial loss of biological function. Ron et al, . / . Biol. Chem. 268: 2984-2988 (1993), incorporated herein by reference in its entirety, reported variant KGF proteins having heparin binding activity even after deleting 3, 8, or 27 amino-terminal amino acid residues. Similarly, interferon gamma exhibited up to ten times higher activity after deleting 8-10 amino acidPCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1 residues from the carboxy terminus of this protein. (Dobeli et al, . / . Biotechnology 7:199-216 (1988), incorporated herein by reference in its entirety.)

[0106] Moreover, ample evidence demonstrates that variants often retain a biological activity similar to that of the naturally occurring protein. For example, Gayle and coworkers ( Biol. Chem 268: 22105-22111 (1993), incorporated herein by reference in its entirety) conducted extensive mutational analysis of human cytokine IL-la. They used random mutagenesis to generate over 3,500 individual IL-la mutants that averaged 2.5 amino acid changes per variant over the entire length of the molecule. Multiple mutations were examined at every possible amino acid position. The investigators found that "[m]ost of the molecule could be altered with little effect on either [binding or biological activity]." (See Abstract.) In fact, only 23 unique amino acid sequences, out of more than 3,500 nucleotide sequences examined, produced a protein that significantly differed in activity from wild-type.

[0107] As stated above, variants or derivatives include, e.g. , modified polypeptides. In some aspects, variants or derivatives of, e.g, polypeptides, polynucleotides, lipids, glycoproteins, are the result of chemical modification and / or endogenous modification. In some aspects, variants or derivatives are the result of in vivo modification. In some aspects, variants or derivatives are the result of in vitro modification. In yet other aspects, variant or derivatives are the result of intracellular modification in producer cells, e.g, T cells.

[0108] Modifications present in variants and derivatives include, e g. , acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation (Mei et al, Blood 116:270-79 (2010), which is incorporated herein by reference in its entirety), proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.

[0109] The term "signaling domain" refers to the functional portion of a protein which acts by transmitting information within the cell to regulate cellular activity via defined signalingPCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1 pathways by generating second messengers or functioning as effectors by responding to such messengers.

[0110] An "intracellular domain," as the term is used herein, refers to an intracellular portion of a molecule. The intracellular signaling domain can generate a signal that promotes an immune effector function of the CAR containing cell, e.g., a CART cell. Examples of immune effector function, e.g, in a CART cell, include cytolytic activity and helper activity, including the secretion of cytokines. In some aspects, the intracellular signal domain is the portion of the protein which transduces the effector function signal and directs the cell to perform a specialized function. While the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion can be used in place of the intact chain as long as it transduces the effector function signal. The term “intracellular signaling domain” is thus meant to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.

[0111] In an embodiment, the intracellular signaling domain can comprise a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from the molecules responsible for primary stimulation, or antigen dependent simulation. In an embodiment, the intracellular signaling domain can comprise a costimulatory intracellular domain. Exemplary costimulatory intracellular signaling domains include those derived from molecules responsible for costimulatory signals, or antigen independent stimulation. For example, in the case of a CAR-T, a primary intracellular signaling domain can comprise a cytoplasmic sequence of a T cell receptor, and a costimulatory intracellular signaling domain can comprise cytoplasmic sequence from co-receptor or costimulatory molecule.

[0112] A primary intracellular signaling domain can comprise a signaling motif which is known as an immunoreceptor tyrosine-based activation motif or IT AM. Examples of IT AM containing primary cytoplasmic signaling sequences include, but are not limited to, those derived from CD3 zeta, FcR gamma, common FcR gamma (FCER1G), Fc gamma Rlla, FcRbeta (Fc Epsilon Rib), CD3 gamma, CD3 delta, CD3 epsilon, CD22, CD79a, CD79b, CD278 (ICOS), FceRI, CD66d, CD32, DAPIO and DAP12.

[0113] The terms "covalently linked," "fused," and grammatical variants thereof are used interchangeably and refer to a first moiety, e.g., a first amino acid sequence or nucleotidePCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1 sequence, covalently or non-covalently joined to a second moiety, e.g, a second amino acid sequence or nucleotide sequence, respectively. The first moiety can be directly joined or juxtaposed to the second moiety or alternatively an intervening moiety can covalently join the first moiety to the second moiety. The term "linked" means not only a fusion of a first moiety to a second moiety at the C-terminus or the N-terminus, but also includes insertion of the whole first moiety (or the second moiety) into any two points, e.g, amino acids, in the second moiety (or the first moiety, respectively). In some aspects, the first moiety is linked to a second moiety by a peptide bond or a linker. The first moiety can be linked to a second moiety by a phosphodiester bond or a linker. The linker can be a peptide or a polypeptide (for polypeptide chains) or a nucleotide or a nucleotide chain (for nucleotide chains) or any chemical moiety (for polypeptide or polynucleotide chains or any chemical molecules).

[0114] As used herein, the term "pharmaceutical composition" refers to one or more of the compounds described herein, such as, e.g. , a CAR of the present disclosure or a cell expressing a CAR of the present disclosure, mixed or intermingled with, or suspended in one or more other chemical components, such as pharmaceutically-acceptable carriers and excipients. One purpose of a pharmaceutical composition is to facilitate administration of preparations of, e.g, cell expressing a CAR of the present disclosure to a subject.

[0115] The terms "excipient" and "carrier" are used interchangeably and refer to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound, e.g, a CAR of the present disclosure.

[0116] The terms "pharmaceutically-acceptable carrier," "pharmaceutically-acceptable excipient," and grammatical variations thereof, encompass any of the agents approved by a regulatory agency of the U.S. Federal government or listed in the U.S. Pharmacopeia for use in animals, including humans, as well as any carrier or diluent that does not cause the production of undesirable physiological effects to a degree that prohibits administration of the composition to a subject and does not abrogate the biological activity and properties of the administered compound. Included are excipients and carriers that are useful in preparing a pharmaceutical composition and are generally safe, non-toxic, and desirable.

[0117] The terms "subject," "patient," "individual," and "host," and variants thereof are used interchangeably herein and refer to any mammalian subject, including without limitation, humans, domestic animals (e.g, dogs, cats and the like), farm animals (e.g., cows, sheep, pigs,PCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1 horses and the like), and laboratory animals ( e.g monkey, rats, mice, rabbits, guinea pigs and the like) for whom diagnosis, treatment, or therapy is desired, particularly humans. The methods described herein are applicable to both human therapy and veterinary applications.

[0118] As used herein, the phrase "subject in need thereof’ includes subjects, such as mammalian subjects, that would benefit from administration of a CAR of the present disclosure, e g., to improve hemostasis.

[0119] The terms "treat," "treatment," or "treating," as used herein refers to, e.g, the reduction in severity of a disease or condition; the reduction in the duration of a disease course; the amelioration or elimination of one or more symptoms associated with a disease or condition; the provision of beneficial effects to a subject with a disease or condition, without necessarily curing the disease or condition. The term also include prophylaxis or prevention of a disease or condition or its symptoms thereof. In some aspects, the term "treating" or "treatment" means inducing an immune response in a subject against an antigen.

[0120] The terms "prevent," "preventing," and variants thereof as used herein, refer partially or completely delaying onset of an disease, disorder and / or condition; partially or completely delaying onset of one or more symptoms, features, or clinical manifestations of a particular disease, disorder, and / or condition; partially or completely delaying onset of one or more symptoms, features, or manifestations of a particular disease, disorder, and / or condition; partially or completely delaying progression from a particular disease, disorder and / or condition; and / or decreasing the risk of developing pathology associated with the disease, disorder, and / or condition. In some aspects, preventing an outcome is achieved through prophylactic treatment.

[0121] As used herein the term "therapeutically effective amount" is the amount of reagent or pharmaceutical compound comprising a CAR of the present disclosure that is sufficient to a produce a desired therapeutic effect, pharmacologic and / or physiologic effect on a subject in need thereof.

[0122] A therapeutically effective amount can be a "prophylactically effective amount" as prophylaxis can be considered therapy. As used herein, "prophylactic" refers to a therapeutic or course of action used to prevent the onset of a disease or condition, or to prevent or delay a symptom associated with a disease or condition. As used herein, a "prophylaxis" refers to a measure taken to maintain health and prevent the onset of a disease or condition, or to prevent or delay a symptom associated with a disease or condition.PCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1

[0123] CARs WITH IgG DERIVED SPACERS

[0124] Of the five immunoglobulin isotypes, immunoglobulin G (IgG) is most abundant in human serum. The four subclasses, IgGl, IgG2, IgG3, and IgG4, which are highly conserved, differ in their constant region, particularly in their hinges and upper CH2 domains. These regions are involved in binding to both IgG-Fc receptors (FcyR) and Clq. As a result, the different subclasses have different effector functions, both in terms of triggering FcyR-expressing cells, resulting in phagocytosis or antibody-dependent cell-mediated cytotoxicity, and activating complement.

[0125] The hinge exon of IgGl encompasses a very flexible hinge. IgG2 has a shorter hinge than IgGl. The lower hinge region of IgG2 (actually encoded by the CH2 region) also has a one amino acid deletion (lacking one of the double Glycines found at position 235-6 of the IgGl lower hinge), resulting in IgG2 having the shortest hinge of all the IgG subclasses. In addition, the hinges of IgG2 are even more rigid due to a poly-proline helix, stabilized by up to four (with some exceptions discussed below) extra inter-heavy chain disulfide bridges. These properties restrict the flexibility of the IgG2 molecule. Similarly, the hinge region of IgG4 (SEQ ID NO: 4453) is also shorter than the IgGl hinge region. The flexibility of the hinge region of IgG4 is intermediate between that of IgGl and IgG2. Unlike the IgG2 hinge, the IgG4 hinge contains the double Glycines found at position 235-6 of the IgGl lower hinge.

[0126] IgG3 has a much longer hinge region than any other IgG subclasses or Ig human isotype, i.e., about four times as long as the IgGl hinge, containing up to 62 amino acids (including 21 prolines and 11 cysteines), forming a poly-proline helix with limited flexibility (SEQ ID NO:2813). The exact length of the hinge varies between allotypes of IgG3, which apparently has undergone much more evolutionary radiation than the other subclasses. In IgG3, the Fab fragments are relatively far away from the Fc fragment, giving the molecule a greater flexibility. This long hinge of IgG3 is a result of duplications of a hinge exon, encoded by one exon in IgGl, IgG2, and IgG4, but up to four exons in IgG3. One of those exons is common to all IgG3 allotypes, but it also has 1-3 copies of a homologous second type of IgG3-hinge exon. The elongated hinge in IgG3 is also responsible for its higher molecular weight compared to the otherPCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1 subclasses. The difference in hinge flexibility influences the relative orientation and movement of the Fab arms and Fc tail of the IgG antibody.

[0127] In IgG2, structural hinge isomers have been observed as a result of alternative formation of disulfide bonds between the cysteines in the hinge region of the heavy chains and those involved in the formation of disulfide bonds between the light and heavy chain. These isomers were found particularly in IgG2 antibodies with kappa-light chains, but much less for lambda light chains. The major forms are the classical A form, with four disulfide bridges between the two IgG2 heavy chains, and the B form in which one hinge cysteine forms a disulfide bond with the light chain. However, other configurations exist, as these isoforms apparently form independent of each other, giving rise to A / A, B / B, but also A / B isoforms

[0128] Two isomers of IgG4 differing in the disulfide bonding of hinge cysteines coexist.

[0129] The core hinge of IgG is formed by a CXXC motif, also found in redox-reactive proteins such as thioredoxins. Compared to IgGl, with a relatively rigid CPPC motif, intra chain disulfide bonds are more easily formed between these cysteines found at positions 226 and 229 in IgG4, which possesses a CPSC core hinge. The result is an observable amount of non-covalently linked half-molecules (consisting of one heavy and one light chain, HL, as opposed to the classical configuration of H2L2) in addition to covalently linked inter-chain isomers. An S228P mutant of IgG4, thus with an IgGLcore hinge, does not form half-molecules, which is in agreement with the finding that this species does not occur in IgGl.

[0130] As used herein, the terms "IgGl hinge", “hinge region of an IgGl” refers to the core plus upper hinge of a IgGl hinge. In some aspects, the IgGl hinge also comprises the lower hinge or a subsequence thereof.

[0131] In some aspects, an IgGl derived CAR spacer comprises consecutive amino acids of the sequence set forth in SEQ ID NO: 6. In some aspects, the IgGl derived CAR spacer comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the sequence set forth in SEQ ID NO: 6.

[0132] In some aspects, a CAR comprising an IgGl derived spacer is capable of inducing an increased interferon-gamma level, e.g., at least 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%,PCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1 at least about 90%, at least about 100%, at least about 120%, at least about 150%, compared to a CAR without an IgGl derived spacer.

[0133] In some aspects, a CAR comprising an IgGl derived spacer is capable of inducing an increased TNF-a level, e.g., at least 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 120%, at least about 150%, compared to a compared to a CAR without an IgGl derived spacer.

[0134] In some aspects, the present disclosure provides an anti-BCMA CAR-expressing cell, e g., CAR-T, e.g, a cell expressing an anti-BCMA CAR construct or encoded by a BCMA binding CAR comprising a scFv, CDRs, or VH and VL chains, wherein the anti-BCMA CAR comprises a CAR spacer of the present disclosure. An anti-BCMA CAR-expressing cell, e.g, CAR-T can be generated by engineering a BCMA CAR that comprises a BCMA binding domain and a CAR spacer of the present disclosure into a cell (e.g, a T cell or NK cell), e.g, for administration to a subject in need thereof.

[0135] In some aspects, the BCMA CAR comprises a sequence set forth in SEQ ID NO:30 in which the spacer (set forth in SEQ ID NO:6) is located between the extracellular binding domain and the transmembrane domain.

[0136] In some aspects, the BCMA CAR comprises a sequence set forth in SEQ ID NO: 32 in which the spacer (set forth in SEQ ID NO:6) is located between the extracellular binding domain and the transmembrane domain.

[0137] In some aspects, the BCMA CAR comprises a sequence set forth in SEQ ID NO: 34 in which the spacer (set forth in SEQ ID NO:6) is located between the extracellular binding domain and the transmembrane domain.

[0138] In some aspects, the present disclosure provides an anti-DLL3 CAR-expressing cell, e.g., CAR-T, e.g, a cell expressing an anti-DLL3 CAR construct or encoded by a DLL3 binding CAR comprising a scFv, CDRs, or VH and VL chains, wherein the anti-DLL3 CAR comprises a CAR spacer of the present disclosure. An anti-DLL3 CAR-expressing cell, e.g, CAR-T can be generated by engineering a DLL3 CAR that comprises a DLL3 binding domain and a CAR spacer of the present disclosure into a cell (e.g, a T cell or NK cell), e.g, for administration to a subject in need thereof.

[0139] In some aspects, the DLL3 CAR comprises a sequence set forth in SEQ ID NO:PCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT118 in which the spacer (set forth in SEQ ID NO:6) is located between the extracellular binding domain and the transmembrane domain.

[0140] In some aspects, the DLL3 CAR comprises a sequence set forth in SEQ ID NO:20 in which the spacer (set forth in SEQ ID NO:6) is located between the extracellular binding domain and the transmembrane domain.

[0141] In some aspects, the DLL3 CAR comprises a sequence set forth in SEQ ID NO:22 in which the spacer (set forth in SEQ ID NO:6) is located between the extracellular binding domain and the transmembrane domain.

[0142] In some aspects, the CAR (e.g., an anti-BCMA CAR or an anti-DLL3 CAR disclosed herein) comprises an optional leader sequence (e.g., an optional leader sequence described herein), an extracellular binding domain, a CAR spacer of the present disclosure (e.g, a human IgGlderived CAR spacer described herein), a transmembrane domain (e.g, transmembrane domain described herein), and an intracellular stimulatory domain (e.g, intracellular stimulatory domain described herein).

[0143] In some aspects, an exemplary CAR construct (an anti-BCMA CAR or an anti-DLL3 CAR disclosed herein) comprises an optional leader sequence (e.g, a leader sequence described herein), an extracellular binding domain, a CAR spacer of the present disclosure (e.g, a hinge or hinge and constant region derived CAR spacer described herein), a transmembrane domain, an intracellular costimulatory domain (e.g, an intracellular costimulatory domain described herein) and an intracellular stimulatory domain.

[0144] In some aspects, the present disclosure encompasses a recombinant nucleic acid construct comprising a nucleic acid molecule encoding a CAR (an anti-BCMA CAR or an anti-DLL3 CAR disclosed herein), wherein the nucleic acid molecule comprises the nucleic acid sequence encoding a binding domain (e.g., a BCMA-binding domain of an anti-BCMA CAR disclosed herein or a DLL3-binding domain of an anti-DLL3 CAR disclosed herein), e.g, that is contiguous with and in the same reading frame as a nucleic acid sequence encoding an intracellular signaling domain. An exemplary intracellular signaling domain that can be used in the CAR includes, but is not limited to, one or more intracellular signaling domains of, e.g, CD3- zeta, CD28, 4- IBB, and the like. In some aspects, the CAR (e.g., an anti-BCMA or an anti-DLL3 CAR disclosed herein) can comprise any combination of CD3-zeta, CD28, 4-1BB, and the like.

[0145] In some aspects, the binding domain (e.g., a BCMA-binding domain in an anti-PCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1BCMA CAR of the present disclosure, or a DLL3-binding domain in an anti-DLL3 CAR disclosed herein) is characterized by particular functional features or properties of an antibody or antigen-binding antibody fragment. For example, in some aspects, the portion of a CAR composition of the disclosure that comprises an antigen-binding domain specifically binds a human antigen or a fragment thereof (e.g., a human BCMA -binding domain in an anti-BCMA CAR of the present disclosure, or a human DLL3 -binding domain in an anti-DLL3 CAR disclosed herein). In certain aspects, the scFv is contiguous with and in the same reading frame as a leader sequence.

[0146] In some aspects, the binding domain (e g., a BCMA-binding domain in an anti-BCMA CAR of the present disclosure, or a DLL3-binding domain in an anti-DLL3 CAR disclosed herein) is a fragment, e.g., a single chain variable fragment (scFv). In some aspects, the binding domain (e.g., a BCMA-binding domain in an anti-BCMA CAR of the present disclosure, or a DLL3-binding domain in an anti-DLL3 CAR disclosed herein) is a Fv, a Fab, a (Fab')2, or a bifunctional (e.g. bi-specific) hybrid antibody (e.g, Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)). In some aspects, the antibodies and fragments thereof of the disclosure, used to generate a CAR of the present disclosure, binds their target protein (e.g., BCMA or DLL3) or a fragment thereof with wild-type or enhanced affinity. In some instances, a human scFv used in a CAR of the present disclosure can be derived from a display library.

[0147] In some aspects, the polypeptide encoding a CAR of the present disclosure, i.e., a CAR comprising a CAR spacer of the present disclosure (i.e., a human IgGl hinge region and an Fc domain), comprises an extracellular binding domain comprising an antibody or an antigenbinding fragment thereof ( e.g ., an scFv) that specifically binds to a tumor antigen.

[0148] In some aspects, the tumor antigen is selected from the group consisting of BCMA, DLL3, HER2, AFP, CD19, TRAC, TCRp, BCMA, CLL-1, CS1, CD38, CD19, TSHR, CD123, CD22, CD30, CD70, CD171, CD33, EGFRvIII, GD2, GD3, PSMA, ROR2, GPC1, GPC2, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL- 13Ra2, mesothelin, IL-1 IRa, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-beta, SSEA-4, CD20, folate receptor alpha, ERBB2 (Her2 / neu), MUC1, MUC16, EGFR, NCAM, prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD 179a, ALK, Poly sialic acid, PLAC1, GloboH, NY-BR-1, UPK2,PCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, L AGE-1 a, MAGE-A1, legumain, HPV E6,E7, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT- 2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin and telomerase, PCTA- 1 / Galectin 8, MelanA / MARTl, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, Androgen receptor, Cyclin Bl, MYCN, RhoC, TRP- 2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP- 4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxyl esterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FC AR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, CD2, CD3e, CD4, CD5, CD7, the extracellular portion of the APRIL protein, and any combinations thereof.

[0149] In some aspects, the extracellular binding domain of a CAR of the present disclosure is an 1g NAR, a Fab, a Fab', a F(ab)'2, a F(ab)'3, an Fv, a single chain variable fragment (scFv), a bis- scFv, a (scFv)2, a minibody, a diabody, a triabody, a tetrabody, an intrabody, a disulfide stabilized Fv protein (dsFv), a unibody, or a nanobody. In some instances, scFvs can be prepared according to method known in the art (see, for example, Bird et ak, (1988) Science 242:423-426 and Huston et ak, (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). ScFv molecules can be produced by linking VH and VL regions together using flexible polypeptide linkers. The scFv molecules comprise a linker ( e.g ., a Ser-Gly linker) with an optimized length and / or amino acid composition. The linker length can greatly affect how the variable regions of a scFv fold and interact. In fact, if a short polypeptide linker is employed (e.g., between 5-10 amino acids) intrachain folding is prevented. Interchain folding is also required to bring the two variable regions together to form a functional epitope binding site. For examples of linker orientation and size see, e.g, Hollinger et ak 1993 Proc Natl Acad. Sci. U.S.A. 90:6444-6448, U.S. Patent Application Publication Nos. 2005 / 0100543, 2005 / 0175606, 2007 / 0014794, and PCT publication Nos. W02006 / 020258 and W02007 / 024715, is incorporated herein by reference.

[0150] An scFv can comprise a linker of, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or more amino acid residues between its VL and VH regions. The linker sequence may comprise any naturally occurring amino acid. In some embodiments, the linker sequence comprises amino acids glycine and serine. In another aspect, the linker sequence comprises sets of glycine and serine repeats such as (Gly4Ser)n, where n is a positive integer equal to or greater than 1. In some aspects, the linker can be (Gly4Ser)4 orPCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1(Gly4Ser)3or any gly-ser rich linker disclosed above. Variation in the linker length may retain or enhance activity, giving rise to superior efficacy in activity studies.

[0151] In some aspects, the amino acid sequence of the extracellular binding domain or other portions or the entire CAR can be modified, e.g, an amino acid sequence described herein can be modified, e.g, by a conservative substitution. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g, lysine, arginine, histidine), acidic side chains (e.g, aspartic acid, glutamic acid), uncharged polar side chains (e.g, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g, threonine, valine, isoleucine) and aromatic side chains (e.g, tyrosine, phenylalanine, tryptophan, histidine).

[0152] In some specific aspects, the tumor antigen is human B cell maturation antigen "BCMA," also known as BCMA, CD269, and TNFRSF17 (UniProt Q02223), which is a member of the tumor necrosis receptor superfamily that is preferentially expressed in differentiated plasma cells. The extracellular domain of human BCMA consists, according to UniProt of amino acids 1-54 (or 5-51). As used herein, "BCMA" includes proteins comprising mutations, e.g, point mutations, fragments, insertions, deletions and splice variants of full length wild-type BCMA. In some aspects the extracellular binding portion of the CAR recognizes and binds an antigen within the extracellular domain of the BCMA protein. In some aspects, the BCMA protein is expressed on a cancer cell.

[0153] In some aspects, anti-BCMA inhibitors, e.g., CARs of the present disclosure or cells expressing those CARs, can be used to treat multiple myeloma.

[0154] In some aspects, a CAR of the present disclosure comprises an anti BCMA antibody or fragment thereof (e.g, single chain variable fragment (scFv)) disclosed in WO2022 / 175255. (for example, generated as described in WO2022 / 175255 incorporated herein by reference). In other aspects, an anti-BCMA CAR of the present disclosure includes an antibody or fragment thereof comprising the VH and / or VL sequences of BCMB519 described WO2022 / 175255, incorporated herein by reference. In one embodiment, the heavy chain of BCMB519 comprises an amino acid sequence of SEQ ID NO: 3 and the light chain of BCMB519 comprises an amino acid sequence of SEQ ID NO: 4.PCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1

[0155] In some aspects, an extracellular binding domain of the present disclosure is capable of cross-competing with an anti-BCMA antibody, e.g, BCMB519 antibody. The BCMB519 antibody sequences are shown in TABLE 1. In some aspects, the extracellular binding domain useful for the present disclosure binds to the same epitope of the BCMB519 antibody.

[0156] In some aspects, the extracellular binding domain of the present disclosure, e.g., scFv, comprises the VH and the VL of the BCMB519 antibody. In some aspects, the BCMB519 scFv is linked to a transmembrane domain by an amino acid sequence derived from a hinge region of a human immunoglobulin G1 isotype (IgGl) and a crystallizable fragment (Fc) of a human IgGl, e.g, SEQ ID NO: 6.

[0157] In some aspects, anti-BCMA antigen-binding antibody fragments (e.g , scFvs) are conjugated or fused to a biologically active molecule, e.g, to form a CAR of the present disclosure ( i.e , a CAR comprising a CAR spacer of the present disclosure, i.e, a human IgGl hinge derived CAR spacer, a human IgGl Fc derived CAR spacer, or a combination thereof) that directs immune cells, e.g, T cells to respond to BCMA-expressing cells.In some specific aspects, the tumor antigen is delta-like canonical Notch Ligand 3 (DLL3) "DLL3," also known as SCDO1 (UniProt Q9NYJ7), which is an inhibitory Notch ligand that is highly expressed in small cell lung cancer and prostate cancer, but minimally expressed in normal tissues. According to UniProt, the extracellular domain of human DLL3 consists of amino acids 27-492, the transmembrane domain consists of amino acids 493-513, and the cytoplasmic domain consists of amino acids 514-618. As used herein, "DLL3" includes proteins comprising mutations, e.g, point mutations, fragments, insertions, deletions and splice variants of full length wild-type DLL3. In some aspects, the extracellular binding portion of the CAR recognizes and binds an epitope within the extracellular domain of the DLL3 protein. In some aspects, the DLL3 protein is expressed on a cancer cell.

[0158] In some aspects, anti-DLL3 inhibitors, e.g, CARs of the present disclosure or cells expressing those CARs, can be used to treat prostate cancer and lung cancer.

[0159] In some aspects, a CAR of the present disclosure comprises an anti DLL3 antibody or fragment thereof (e g, single chain variable fragment (scFv)) disclosed in WO2022 / 084915. (for example, generated as described in WO2022 / 084915 incorporated herein by reference). In other aspects, an anti-DLL3 CAR of the present disclosure includes an antibody or fragment thereof comprising the VH and / or VL sequences of DL3469. In one embodiment, the heavy chain ofPCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1DL3469 comprises an amino acid sequence of SEQ ID NO: 1 and the light chain of DL3469 comprises an amino acid sequence of SEQ ID NO: 2.

[0160] In some aspects, an extracellular binding domain of the present disclosure is capable of cross-competing with an anti-DLL3 antibody, e.g, DL3469 antibody. The DL3469 antibody sequences are shown in TABLE 1. In some aspects, the extracellular binding domain useful for the present disclosure binds to the same epitope of the DL3469 antibody.

[0161] In some aspects, the extracellular binding domain of the present disclosure, e.g., scFv, comprises the VH and the VL of the DL3469 antibody. In some aspects, the DL3469 scFv is linked to a transmembrane domain by an amino acid sequence derived from a hinge region of a human immunoglobulin G1 isotype (IgGl) and a crystallizable fragment (Fc) of a human IgGl, e g., SEQ ID NO: 6.

[0162] In some aspects, anti-DLL3 antigen-binding antibody fragments ( e.g ., scFvs) are conjugated or fused to a biologically active molecule, e.g., to form a CAR of the present disclosure (i.e., a CAR comprising a CAR spacer of the present disclosure, i.e., a human IgGl hinge derived CAR spacer and a human IgGl Fc derived CAR spacer) that directs immune cells, e g, T cells to respond to DLL3 -expressing cells.

[0163] SIGNALING, TRANSMEMBRANE, COSTIMULATORY DOMAINS

[0164] In some aspects, the intracellular domain of a CAR of the present disclosure (i.e., CAR comprising a CAR spacer of the present disclosure, i.e., a human IgGl hinge and a human IgGl Fc derived CAR spacer) is a signaling domain derived from CD3zeta, FcR gamma, FcRbeta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In some aspect, the CAR further comprises a co-stimulatory domain derived from 2B4, HVEM, ICOS, LAG3, DAPIO, DAP 12, CD27, CD28, 4-1BB (CD137), 0X40 (CD134), CD30, CD40, ICOS (CD278), glucocorticoid-induced tumor necrosis factor receptor (GITR), lymphocyte function-associated antigen- 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, or B7-H3. In some aspects, the CAR further comprises a 4-1 IBB costimulatory domain. In some aspects, the 4-1BB costimulatory domain comprises SEQ ID NO: 9.

[0165] In some aspects, the CAR of the present disclosure (i.e., CAR comprising a CAR spacer of the present disclosure, i.e., a human IgGl hinge derived CAR spacer and a human IgGl FcPCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1 derived CAR spacer) comprises a transmembrane domain of a protein selected from the group consisting of the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD 154. The transmembrane domain may be derived either from a natural or from a recombinant source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. In some aspects, the transmembrane domain is capable of signaling to the intracellular domain(s) whenever the CAR of the present disclosure has bound to a target.

[0166] In some aspects, a transmembrane domain can include at least the transmembrane region(s) of, e.g., KIRDS2, 0X40, CD2, CD27, LFA-1 (CD 11 a, CD18), ICOS (CD278), 4-1BB (CD 137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2Rbeta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD 103, ITGAL, CD1 la, LFA-1, ITGAM, CD1 lb, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD 160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, PAG / Cbp, NKG2D, NKG2C, or CD 19.

[0167] In some aspects, the CAR of the present disclosure (i.e., CAR comprising a CAR spacer of the present disclosure, i.e., a human IgGl hinge and a human IgGl Fc derived CAR spacer) further comprises a sequence encoding a costimulatory domain, e.g, a costimulatory domain described herein. In some aspects, the costimulatory domain comprises a functional signaling domain of a protein selected from the group consisting of 0X40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD1 la / CD18), ICOS (CD278), and 4-1BB (CD137). In some aspects, the costimulatory domain comprises a functional signaling domain of a protein selected from the group consisting of MHC class I molecule, TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, 0X40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CDlla / CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f,PCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1ITGAD, CD1 Id, ITGAE, CD 103, IT GAL, CD1 la, LFA-1, ITGAM, CD1 lb, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD 18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD 160 (BY55), PSGL1, CD 100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP- 76, PAG / Cbp, CD 19a, and a ligand that specifically binds with CD83. In some aspects, the costimulatory domain comprises 4-1BB, CD27, CD28, or ICOS.

[0168] In some aspects, the CAR of the present disclosure (i.e., CAR comprising a CAR spacer of the present disclosure, i.e a human IgGl hinge and a human IgGl Fc derived CAR spacer) further comprises a sequence encoding an intracellular signaling domain, e.g., an intracellular domain described herein. In some aspects, the intracellular domain comprises a functional signaling domain of 4-IBB and / or a functional signaling domain of CD3 zeta. In some aspects, the intracellular domain comprises a functional signaling domain of CD27 and / or a functional signaling domain of CD3 zeta.

[0169] In some aspects, the CAR of the present disclosure (i.e., CAR comprising a CAR spacer of the present disclosure, i.e a human IgGl hinge and a human IgGl Fc derived CAR spacer) further comprises a leader sequence. In some aspects, the intracellular domain comprises CD3z comprising SEQ ID NO: 10.

[0170] In some aspects, the CAR of the present disclosure (i.e., CAR comprising a CAR spacer of the present disclosure, i.e., a human IgGl hinge and a human IgGl Fc derived CAR spacer) comprises an optional leader sequence (e.g, an optional leader sequence described herein), an extracellular binding domain, a CAR spacer of the present disclosure, a transmembrane domain (e.g, transmembrane domain described herein), and an intracellular domain (e.g, intracellular domain described herein).

[0171] In some aspects, an exemplary CAR construct of the present disclosure comprises an optional leader sequence, an extracellular binding domain, a spacer, a transmembrane domain, an costimulatory domain (e.g, a constimulatory domain described herein) and an intracellular stimulatory domain.

[0172] In some aspects, the present disclosure provides a CAR comprising: (i) a BCMA.A scFv comprising SEQ ID NO: 14; (ii) a GGSEGKSSGSGSESKSTGGS linker (SEQ ID NO: 38), (iii) a spacer comprising or consisting of SEQ ID NO: 6, (iv) a transmembrane domain comprisingPCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1SEQ ID NO: 8 and optionally (v) a 4-1BB costimulatory domain comprising SEQ ID NO: 9. In some aspects, the CAR further comprises (vi) a CD3zeta signaling domain comprising SEQ ID NO: 10 or any combination thereof.

[0173] In some aspects, the present disclosure provides a CAR comprising: (i) a BCMA.B scFv comprising SEQ ID NO: 15; (ii) a GGSEGKSSGSGSESKSTGGS linker (SEQ ID NO: 38), (iii) a spacer comprising or consisting of SEQ ID NO: 6, (iv) a transmembrane domain comprising SEQ ID NO: 8 and optionally (v) a 4-1BB costimulatory domain comprising SEQ ID NO: 9. In some aspects, the CAR further comprises (vi) a CD3zeta signaling domain comprising SEQ ID NO: 10 or any combination thereof.

[0174] In some aspects, the present disclosure provides a CAR comprising: (i) a BCMA.C scFv comprising SEQ ID NO: 16; (ii) a GGSEGKSSGSGSESKSTGGS linker (SEQ ID NO: 38), (iii) a spacer comprising or consisting of SEQ ID NO: 6, (iv) a transmembrane domain comprising SEQ ID NO: 8 and optionally (v) a 4-1BB costimulatory domain comprising SEQ ID NO: 9. In some aspects, the CAR further comprises (vi) a CD3zeta signaling domain comprising SEQ ID NO: 10 or any combination thereof.

[0175] In some aspects, the present disclosure provides a CAR comprising: (i) a DLL3.A scFv comprising SEQ ID NO: 11; (ii) a GGSEGKSSGSGSESKSTGGS linker (SEQ ID NO: 38), (iii) a spacer comprising or consisting of SEQ ID NO: 6, (iv) a transmembrane domain comprising SEQ ID NO: 8 and optionally (v) a 4-1BB costimulatory domain comprising SEQ ID NO: 9. In some aspects, the CAR further comprises (vi) a CD3zeta signaling domain comprising SEQ ID NO: 10 or any combination thereof.

[0176] In some aspects, the present disclosure provides a CAR comprising: (i) a DLL3.B scFv comprising SEQ ID NO: 12; (ii) a GGSEGKSSGSGSESKSTGGS linker (SEQ ID NO: 38), (iii) a spacer comprising or consisting of SEQ ID NO: 6, (iv) a transmembrane domain comprising SEQ ID NO: 8 and optionally (v) a 4-1BB costimulatory domain comprising SEQ ID NO: 9. In some aspects, the CAR further comprises (vi) a CD3zeta signaling domain comprising SEQ ID NO: 10 or any combination thereof.

[0177] In some aspects, the present disclosure provides a CAR comprising: (i) a DLL3.C scFv comprising SEQ ID NO: 13; (ii) a GGSEGKSSGSGSESKSTGGS linker (SEQ ID NO: 38), (iii) a spacer comprising or consisting of SEQ ID NO: 6, (iv) a transmembrane domain comprising SEQ ID NO: 8 and optionally (v) a 4-1BB costimulatory domain comprising SEQ ID NO: 9. InPCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1 some aspects, the CAR further comprises (vi) a CD3zeta signaling domain comprising SEQ ID NO: 10 or any combination thereof.

[0178] In some aspects, the present disclosure provides a CAR comprising: (i) a DLL3.D scFv comprising SEQ ID NO: 39; (ii) a GGSEGKSSGSGSESKSTGGS linker (SEQ ID NO: 38), (iii) a spacer comprising or consisting of SEQ ID NO: 6, (iv) a transmembrane domain comprising SEQ ID NO: 8 and optionally (v) a 4-1BB costimulatory domain comprising SEQ ID NO: 9. In some aspects, the CAR further comprises (vi) a CD3zeta signaling domain comprising SEQ ID NO: 10 or any combination thereof.

[0179] In some aspects, the present disclosure provides a polynucleotide sequence encoding a CAR comprising, consisting, or consisting essentially of a protein sequence set forth in SEQ ID NOS: 17 to 37.

[0180] VECTORS

[0181] The present disclosure also provides a vector comprising a polynucleotide encoding a CAR of the present disclosure (i.e., CAR comprising a CAR spacer of the present disclosure, i.e., a human IgGl hinge and a human IgGl Fc derived CAR spacer) operably linked to a regulatory element. In some aspects, the polynucleotide encoding a CAR of the present disclosure is a DNA molecule, or a RNA molecule.

[0182] In some aspects, the vector is a transfer vector. The term "transfer vector" refers to a composition of matter which comprises an isolated nucleic acid (e.g, a polynucleotide of the present disclosure) and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "transfer vector" includes an autonomously replicating plasmid or a virus. The term should also be construed to further include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, a polylysine compound, liposome, and the like. Examples of viral transfer vectors include, but are not limited to, adenoviral vectors, adeno- associated virus vectors, retroviral vectors, lentiviral vectors, and the like.PCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1

[0183] In some aspects, the vector is an expression vector. The term "expression vector" refers to a vector comprising a recombinant polynucleotide ( e.g ., a polypeptide of the present disclosure) comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g, lentiviruses, retroviruses, adenoviruses, and adeno- associated viruses) that incorporate the recombinant polynucleotide.

[0184] In some aspects, the vector is a viral vector, a mammalian vector, or bacterial vector. In some aspects, the vector is selected from the group consisting of an adenoviral vector, a lentivirus, a Sendai virus vector, a baculoviral vector, an Epstein Barr viral vector, a papovaviral vector, a vaccinia viral vector, a herpes simplex viral vector, a hybrid vector, and an AAV vector.

[0185] In some aspects, the adenoviral vector is a third-generation adenoviral vector. ADEASY™ is by far the most popular method for creating adenoviral vector constructs. The system consists of two types of plasmids: shuttle (or transfer) vectors and adenoviral vectors. The transgene of interest is cloned into the shuttle vector, verified, and linearized with the restriction enzyme Pmel. This construct is then transformed into ADEASIER-1 cells, which are BJ5183 E. coli cells containing PADEASY™. PADEASY™ is a ~33Kb adenoviral plasmid containing the adenoviral genes necessary for virus production. The shuttle vector and the adenoviral plasmid have matching left and right homology arms which facilitate homologous recombination of the transgene into the adenoviral plasmid. One can also co-transform standard BJ5183 with supercoiled PADEASY™ and the shuttle vector, but this method results in a higher background of non-recombinant adenoviral plasmids. Recombinant adenoviral plasmids are then verified for size and proper restriction digest patterns to determine that the transgene has been inserted into the adenoviral plasmid, and that other patterns of recombination have not occurred. Once verified, the recombinant plasmid is linearized with Pad to create a linear dsDNA construct flanked by ITRs. 293 or 911 cells are transfected with the linearized construct, and virus can be harvested about 7- 10 days later. In addition to this method, other methods for creating adenoviral vector constructs known in the art at the time the present application was filed can be used to practice the methods disclosed herein.PCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1

[0186] In other aspects, the viral vector is a retroviral vector, e.g., a lentiviral vector (e.g, a third or fourth generation lentiviral vector). The term "lentivirus" refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses. The term "lentiviral vector" refers to a vector derived from at least a portion of a lentivirus genome, including especially a self-inactivating lentiviral vector as provided in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009). Other examples of lentivirus vectors that may be used in the clinic, include but are not limited to, e.g, the LENTIVECTOR® gene delivery technology from Oxford BioMedica, the LENTIMAX™ vector system from Lentigen and the like. Nonclinical types of lentiviral vectors are also available and would be known to one skilled in the art.

[0187] Lentiviral vectors are usually created in a transient transfection system in which a cell line is transfected with three separate plasmid expression systems. These include the transfer vector plasmid (portions of the HIV provirus), the packaging plasmid or construct, and a plasmid with the heterologous envelop gene (env) of a different virus. The three plasmid components of the vector are put into a packaging cell which is then inserted into the HIV shell. The virus portions of the vector contain insert sequences so that the virus cannot replicate inside the cell system. Current third generation lentiviral vectors encode only three of the nine HIV-1 proteins (Gag, Pol, Rev), which are expressed from separate plasmids to avoid recombination-mediated generation of a replication-competent virus. In fourth generation lentiviral vectors, the retroviral genome has been further reduced (see, e.g, TAKARA® LENTI-X™ fourth-generation packaging systems).

[0188] In some aspects, the present disclosure provides a lentiviral vector comprising a polynucleotide sequence encoding: (i) a BCMA.A scFv comprising SEQ ID NO: 14; (ii) a GGSEGKSSGSGSESKSTGGS linker (SEQ ID NO: 38), (iii) a spacer comprising or consisting of SEQ ID NO: 6, (iv) a transmembrane domain comprising SEQ ID NO: 8 and optionally (v) a 4-1BB costimulatory domain comprising SEQ ID NO: 9. In some aspects, the lentiviral vector further comprises (vi) a CD3zeta signaling domain comprising SEQ ID NO: 10 or any combination thereof.PCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1

[0189] In some aspects, the present disclosure provides a lentiviral vector comprising a polynucleotide sequence encoding: (i) a BCMA.B scFv comprising SEQ ID NO: 15; (ii) a GGSEGKSSGSGSESKSTGGS linker (SEQ ID NO: 38), (iii) a spacer comprising or consisting of SEQ ID NO: 6, (iv) a transmembrane domain comprising SEQ ID NO: 8 and optionally (v) a 4-1BB costimulatory domain comprising SEQ ID NO: 9. In some aspects, the lentiviral vector further comprises (vi) a CD3zeta signaling domain comprising SEQ ID NO: 10 or any combination thereof.

[0190] In some aspects, the present disclosure provides a lentiviral vector comprising a polynucleotide sequence encoding: (i) a BCMA.C scFv comprising SEQ ID NO: 16; (ii) a GGSEGKSSGSGSESKSTGGS linker (SEQ ID NO: 38), (iii) a spacer comprising or consisting of SEQ ID NO: 6, (iv) a transmembrane domain comprising SEQ ID NO: 8 and optionally (v) a 4-1BB costimulatory domain comprising SEQ ID NO: 9. In some aspects, the lentiviral vector further comprises (vi) a CD3zeta signaling domain comprising SEQ ID NO: 10 or any combination thereof.

[0191] In some aspects, the present disclosure provides a lentiviral vector comprising a polynucleotide sequence encoding: (i) a DLL3.A scFv comprising SEQ ID NO: 11; (ii) a GGSEGKSSGSGSESKSTGGS linker (SEQ ID NO: 38), (iii) a spacer comprising or consisting of SEQ ID NO: 6, (iv) a transmembrane domain comprising SEQ ID NO: 8 and optionally (v) a 4-1BB costimulatory domain comprising SEQ ID NO: 9. In some aspects, the lentiviral vector further comprises (vi) a CD3zeta signaling domain comprising SEQ ID NO: 10 or any combination thereof.

[0192] In some aspects, the present disclosure provides a lentiviral vector comprising a polynucleotide sequence encoding: (i) a DLL3.B scFv comprising SEQ ID NO: 12; (ii) a GGSEGKSSGSGSESKSTGGS linker (SEQ ID NO: 38), (iii) a spacer comprising or consisting of SEQ ID NO: 6, (iv) a transmembrane domain comprising SEQ ID NO: 8 and optionally (v) a 4-1BB costimulatory domain comprising SEQ ID NO: 9. In some aspects, the lentiviral vector further comprises (vi) a CD3zeta signaling domain comprising SEQ ID NO: 10 or any combination thereof.

[0193] In some aspects, the present disclosure provides a lentiviral vector comprising a polynucleotide sequence encoding: (i) a DLL3.C scFv comprising SEQ ID NO: 13; (ii) a GGSEGKSSGSGSESKSTGGS linker (SEQ ID NO: 38), (iii) a spacer comprising or consistingPCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1 of SEQ ID NO: 6, (iv) a transmembrane domain comprising SEQ ID NO: 8 and optionally (v) a 4-1BB costimulatory domain comprising SEQ ID NO: 9. In some aspects, the lentiviral vector further comprises (vi) a CD3zeta signaling domain comprising SEQ ID NO: 10 or any combination thereof.

[0194] In some aspects, the present disclosure provides a lentiviral vector comprising a polynucleotide sequence encoding: (i) a DLL3.D scFv comprising SEQ ID NO: 39; (ii) a GGSEGKSSGSGSESKSTGGS linker (SEQ ID NO: 38), (iii) a spacer comprising or consisting of SEQ ID NO: 6, (iv) a transmembrane domain comprising SEQ ID NO: 8 and optionally (v) a 4-1BB costimulatory domain comprising SEQ ID NO: 9. In some aspects, the lentiviral vector further comprises (vi) a CD3zeta signaling domain comprising SEQ ID NO: 10 or any combination thereof.

[0195] In some aspects, non-viral methods can be used to deliver a nucleic acid comprising a polynucleotide encoding a CAR of the present disclosure into a cell or tissue of a subject. In some aspects, the non-viral method includes the use of a transposon. In some aspects, use of a non-viral method of delivery permits reprogramming of cells, e.g., T or NK cells, and direct infusion of the cells into the subject. In some aspects, a nucleic acid sequence comprising a polynucleotide encoding a CAR of the present disclosure can be inserted into the genome of a target cell (e.g, a T cell) or a host cell (e.g, a cell for recombinant expression of the CAR polypeptide) by using CRISPR / Cas systems and genome edition alternatives such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and meganucleases (MNs).

[0622] In some, a CAR of the present disclosure (i.e., CAR comprising a CAR spacer of the present disclosure, i.e., a hinge derived CAR spacer, a loop derived CAR spacer, or a combination thereof) can be expressed in a cell using bicistronic or multi cistronic expression vectors. In some aspects, bicistronic or multi cistronic vectors include, but not limited to, (1) multiple promoters fused to multiple CARs' open reading frames; (2) insertion of splicing signals between units of a CAR; fusion of CARs of which expressions are driven by a single promoter;(3) insertion of proteolytic cleavage sites between units of CAR (self-cleavage peptide); and (iv) insertion of internal ribosomal entry sites (IRESs).

[0196] In some aspects, multiple CAR units are expressed in a single open reading frame (ORF), thereby creating a single polypeptide having multiple CAR units, wherein at least one of the CARs is a CAR of the present disclosure. In some aspects, an amino acid sequence or linkerPCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1 containing a high efficiency cleavage site is disposed between each CAR unit. As used herein, high cleavage efficiency is defined as more than 50 %, more than 70 %, more than 80%, or more than 90% of the translated protein is cleaved. Cleavage efficiency can be measured by Western Blot analysis.

[0197] Non-limiting examples of high efficiency cleavage sites include porcine teschovirus-1 2A (P2A), FMDV 2A (abbreviated herein as F2A); equine rhinitis A virus (ERAV) 2A (E2A); and Thoseaasigna virus 2A (T2A), cytoplasmic polyhedrosis virus 2A (BmCPV2A) and flacherie Virus 2A (BmIFV2A), or a combination thereof. In some aspects, the high efficiency cleavage site is P2A. High efficiency cleavage sites are described in Kim et al. (2011) High Cleavage Efficiency of a 2A Peptide Derived from Porcine Teschovirus-1 in Human Cell Lines, Zebrafish and Mice. PLoS ONE 6(4): el8556, the contents of which are incorporated herein by reference.

[0198] In some aspects, multiple CAR units are expressed in a single open reading frame (ORF), expression is under the control of a strong promoter.

[0199] In some aspects, a polynucleotide encoding a CAR of the present disclosure (i.e., CAR comprising a CAR spacer of the present disclosure, i.e a human IgGl hinge derived CAR spacer and a human IgGl Fc derived CAR spacer) can comprise at least one chemically modified nucleobase, sugar, backbone, or any combination thereof. Thus, a polynucleotide encoding a CAR of the present disclosure can comprise one or more modifications. In some aspects, a polynucleotide encoding a CAR of the present disclosure comprises at least one nucleotide analogue. In some aspects, at least one nucleotide analogue introduced by using IVT (in vitro transcription) or chemical synthesis is selected from the group consisting of a 2'-0-methoxyethyl- RNA (2'-MOE-RNA) monomer, a 2'-fluoro-DNA monomer, a 2'-0-alkyl-RNA monomer, a 2'- amino-DNA monomer, a locked nucleic acid (LNA) monomer, a cEt monomer, a cMOE monomer, a 5'-Me-LNA monomer, a 2'-(3 -hydroxy )propyl-RNA monomer, an arabino nucleic acid (ANA) monomer, a 2'-fluoro-ANA monomer, an anhydrohexitol nucleic acid (HNA) monomer, an intercalating nucleic acid (INA) monomer, and a combination of two or more of said nucleotide analogues. In some aspects, the optimized nucleic acid molecule comprises at least one backbone modification, for example, a phosphorothioate internucleotide linkage.

[0200] In some aspects, a polynucleotide encoding a CAR of the present disclosure can be chemically modified at terminal locations, for example by introducing M (2 '-O-methyl), MS (2 -PCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1O-methyl 3' phosphorothioate), or MSP (2'-0-methy 3 ’thioPACE, phosphonoacetate) modifications, or combinations thereof at positions 1, 2, 3 respect to the 5’ and / or 3’ termini.

[0201] Modified polynucleotides encoding a CAR of the present disclosure (i.e., CAR comprising a CAR spacer of the present disclosure, i.e., a human IgGl hinge derived CAR spacer, a human IgGl Fc derived CAR spacer, or a combination thereof) need not be uniformly modified along the entire length of the molecule. Different nucleotide modifications and / or backbone structures may exist at various positions in the nucleic acid. One of ordinary skill in the art will appreciate that the nucleotide analogs or other modification(s) may be located at any position(s) of a nucleic acid such that the function of the nucleic acid is not substantially decreased. A modification may also be a 5' or 3' terminal modification. The nucleic acids may contain at a minimum one and at maximum 100% modified nucleotides, or any intervening percentage, such as at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% modified nucleotides.

[0630] In some aspects, a polynucleotide encoding a CAR of the present disclosure (i.e.,

[0202] CAR comprising a CAR spacer of the present disclosure, i.e., a human IgGl hinge and a human IgGl Fc derived CAR spacer) can include modifications to prevent rapid degradation by endo- and exo-nucleases. Modifications include, but are not limited to, for example, (a) end modifications, e.g., 5' end modifications (phosphorylation dephosphorylation, conjugation, inverted linkages, etc.), 3' end modifications (conjugation, DNA nucleotides, inverted linkages, etc ), (b) base modifications, e.g, replacement with modified bases, stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, or conjugated bases, (c) sugar modifications (e.g, at the 2' position or 4' position) or replacement of the sugar, as well as (d) internucleoside linkage modifications, including modification or replacement of the phosphodiester linkages.

[0203] Specific examples of synthetic, modified polynucleotides encoding a CAR of the present disclosure (i.e., CAR comprising a CAR spacer of the present disclosure, i.e., a human IgGl hinge and a human IgGl Fc derived CAR spacer) useful with the methods described herein include, but are not limited to, polynucleotides encoding a CAR of the present disclosurePCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1 containing modified or non-natural intemucleoside linkages. Synthetic, modified polynucleotides encoding a CAR of the present disclosure having modified intemucleoside linkages include, among others, those that do not have a phosphorus atom in the intemucleoside linkage. In some aspects, a synthetic, modified polynucleotide encoding a CAR of the present disclosure has a phosphorus atom in its intemucleoside linkage(s).

[0204] Non-limiting examples of modified intemucleoside linkages include phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, T-5' linked analogs of these, and those) having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or T-5' to 5'-T. Various salts, mixed salts and free acid forms are also included.

[0205] Modified intemucleoside linkages that do not include a phosphorus atom therein have intemucleoside linkages that are formed by short chain alkyl or cycloalkyl intemucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl intemucleoside linkages, or one or more short chain heteroatomic or heterocyclic intemucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, 0, S and CH2 component parts.

[0206] I n some aspects, a polynucleotide encoding a CAR of the present disclosure (i.e., CAR comprising a CAR spacer of the present disclosure, i.e., a human IgGl hinge derived CAR spacer, a human IgGl Fc derived CAR spacer, or a combination thereof) can be codon optimized by introducing one or more synonymous codon changes. As used herein, the terms "codon optimization," "codon optimized," and grammatical variants thereof refer to the modification of the primary sequence of a nucleic acid by replacing synonymous codons in order to increase its translational efficiency. Accordingly, codon optimization comprises switching the codons used in a polynucleotide encoding a CAR of the present disclosure without changing the amino acidPCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1 sequence that it encodes for, which typically dramatically increases the abundance of the protein the codon optimized gene encodes because it generally removes "rare" codons and replaces them with abundant codons, or removes codon with a low tRNA recharge rate with codon with high tRNA recharge rates. Such codon optimization can, for example, (i) improve protein yield in recombinant protein expression, or (ii) improve the stability, half life, or other desirable property of an mRNA or a DNA encoding a binding molecule disclosed herein, wherein such mRNA or DNA is administered to a subject in need thereof.

[0207] The sequences of polynucleotides encoding a CAR of the present disclosure (i.e., CAR comprising a CAR spacer of the present disclosure, i.e., a human IgGl hinge derived CAR spacer and a human IgGl Fc derived CAR spacer) can be codon optimized using any methods known in the art at the time the present application was filed.

[0208] In some aspects, a polynucleotide encoding a CAR of the present disclosure (i.e., CAR comprising a CAR spacer of the present disclosure, i.e., a human IgGl hinge and a human IgGl Fc derived CAR spacer) has been sequence optimized. As used herein, the term "sequence optimized" refers to the modification of the sequence of a nucleic acid by to introduce features that increase its translational efficiency, remove features that reduce its translational efficiency, or in general improve properties related to expression efficacy after administration in vivo. Such properties include, but are not limited to, improving nucleic acid stability (e g mRNA stability), increasing translation efficacy in the target tissue, reducing the number of truncated proteins expressed, improving the folding or prevent misfolding of the expressed proteins, reducing toxicity of the expressed products, reducing cell death caused by the expressed products, or increasing and / or decreasing protein aggregation.

[0209] The present disclosure contemplates modifications to the entire CAR construct e.g., modifications in one or more amino acid sequences of the various domains of the CAR construct in order to generate functionally equivalent molecules. The CAR construct can be modified to retain at least about 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% sequence identity of the starting CAR construct. The present disclosure also contemplates modifications of specific regions of a CAR, e.g, modifications in one or more amino acid sequences of one or more CDRs of a CAR construct in order to generate functionally equivalent molecules.PCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1

[0210] CELLS

[0211] The present disclosure also provides a genetically modified cell comprising a polynucleotide encoding a CAR of the present disclosure (i.e., CAR comprising a CAR spacer of the present disclosure, i.e., a human IgGl hinge and a human IgGl Fc derived CAR spacer). In some aspects, the CAR is recombinantly expressed by a cell genetically modified to express a CAR, wherein the cell comprises by one or more of the polynucleotide sequences or the vectors encoding a CAR of the present disclosure.

[0212] In some aspects, the genetically modified cell disclosed herein has been transfected with a polynucleotide or vector encoding a CAR of the present disclosure. The term "transfected" (or equivalent terms "transformed" and "transduced") refers to a process by which exogenous nucleic acid, e.g, a polynucleotide or vector encoding a CAR of the present disclosure, is transferred or introduced into the genome of the host cell, e g, a T cell. A "transfected" cell is one which has been transfected, transformed or transduced with exogenous nucleic acid, e.g, a polynucleotide or vector encoding a CAR of the present disclosure. The cell includes the primary subject cell and its progeny.

[0213] In some aspects, the cell (e.g, T cell) is transfected with a vector of the present disclosure, e.g, an AAV vector or a lentiviral vector. In some such aspects, the cell may stably express the CAR of the present disclosure.

[0214] In some aspects, the cell (e.g, T cell) is transfected with a nucleic acid, e.g, mRNA, cDNA, DNA, encoding a CAR of the present disclosure. In some such aspects, the cell may transiently express the CAR of the present disclosure. For example, an RNA construct can be directly transfected into a cell. A method for generating mRNA for use in transfection involves in vitro transcription (IVT) of a template with specially designed primers, followed by polyA addition, to produce a construct containing 3' and 5' untranslated sequence (UTR), a 5' cap and / or Internal Ribosome Entry Site (IRES), the nucleic acid to be expressed, and a polyA tail, typically 50-2000 bases in length. RNA so produced can efficiently transfect different kinds of cells. In some aspects, the template includes sequences for the CAR of the present disclosure. In an aspect, an RNA CAR vector is transduced into a T cell by electroporation.PCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1

[0215] In some aspects, the cell is an immune effector cell. As used herein, term "immune effector cell" refers to a cell that is involved in an immune response, e.g., in the promotion of an immune effector response. "Immune effector function" or "immune effector response," refer to function or response, e.g, of an immune effector cell, that enhances or promotes an immune attack of a target cell. E.g., an immune effector function or response refers a property of a T or NK cell that promotes killing or the inhibition of growth or proliferation, of a target cell. In the case of a T cell, primary stimulation and co-stimulation are examples of immune effector function or response.

[0216] The term "effector function" refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines. The intracellular signaling domain of a CAR can generate a signal that promotes an immune effector function of the CAR containing cell, e.g., a CAR-T cell. Examples of immune effector function, e.g, in a CAR-T cell, include cytolytic activity and helper activity, including the secretion of cytokines. In some aspects, the intracellular signal domain is the portion of the CAR which transduces the effector function signal and directs the cell to perform a specialized function. While the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. The term intracellular signaling domain is thus meant to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.

[0217] In some aspects, the intracellular domain can comprise a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from the molecules responsible for primary stimulation, or antigen dependent simulation. In some aspects, the intracellular signaling domain can comprise a costimulatory intracellular domain. Exemplary costimulatory intracellular signaling domains include those derived from molecules responsible for costimulatory signals, or antigen independent stimulation. For example, in the case of a CAR-T, a primary intracellular signaling domain can comprise a cytoplasmic sequence of a T cell receptor, and a costimulatory intracellular signaling domain can comprise cytoplasmic sequence from co-receptor or costimulatory molecule.PCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1

[0218] A primary intracellular signaling domain can comprise a signaling motif which is known as an immunoreceptor tyrosine-based activation motif or IT AM. Examples of IT AM containing primary cytoplasmic signaling sequences include, but are not limited to, those derived from CD3 zeta, FcR gamma, common FcR gamma (FCER1G), Fc gamma Rlla, FcRbeta (Fc Epsilon Rib), CD3 gamma, CD3 delta, CD3 epsilon, CD22, CD79a, CD79b, CD278 (“ICOS”), FceRI, CD66d, CD32, DAPIO and DAP12.

[0219] Examples of immune effector cells include, e.g., T cells, e.g, alpha / beta T cells and gamma / delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid-derived phagocytes. Innate lymphoid cells (ILCs) are a group of innate immune cells that are derived from common lymphoid progenitor (CLP) and belong to the lymphoid lineage. These cells are defined by absence of antigen specific B or T cell receptor because of the lack of recombination activating gene (RAG). ILCs do not express myeloid or dendritic cell markers. ILCs has varying physiological functions; some functions are analogous to helper T cells, while the group also includes cytotoxic NK cells. Accordingly, in some aspects, the cell genetically modified to express a CAR of the present disclosure is, e.g, a T cell, an NK cell, an NKT cell, or an ILC cell.

[0220] T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors.

[0221] PHARMACEUTICAL COMPOSITIONS

[0222] The present disclosure also provides pharmaceutical compositions comprising compositions disclosed herein, e.g, a polynucleotide encoding a CAR of the present disclosure, a vector comprising a polynucleotide encoding a CAR of the present disclosure, or a genetically modified cell comprising a polynucleotide or a vector encoding a CAR of the present disclosure, which are suitable for administration to a subject.

[0223] The pharmaceutical compositions generally comprise polynucleotide, vector, or cell encoding or comprising a CAR of the present disclosure and a pharmaceutically-acceptable excipient or carrier in a form suitable for administration to a subject. PharmaceuticallyPCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1 acceptable excipients or carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition.

[0224] There is a wide variety of suitable formulations of pharmaceutical compositions comprising a CAR of the present disclosure (see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 18th ed. (1990)). The pharmaceutical compositions are generally formulated sterile and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[0225] In certain aspects, the pharmaceutical composition is co-administered with of one or more additional therapeutic agents, in a pharmaceutically acceptable carrier. In some aspects, the pharmaceutical composition comprising the CAR of the present disclosure is administered prior to administration of the additional therapeutic agent(s). In other aspects, the pharmaceutical composition comprising the CAR of the present disclosure is administered after the administration of the additional therapeutic agent(s). In further aspects, the pharmaceutical composition comprising the CAR of the present disclosure is administered concurrently with the additional therapeutic agent(s).

[0226] Acceptable carriers, excipients, or stabilizers are nontoxic to recipients (e.g, animals or humans) at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, di saccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g, Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

[0227] Examples of carriers or diluents include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin. The use of such media and compounds for pharmaceutically active substances is well known in the art. Except insofar asPCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1 any conventional media or compound is incompatible with the compositions of the present disclosure (e.g, polynucleotides, vectors, or cells), use thereof in the compositions is contemplated. VI. Libraries and methods of use.

[0228] INDICATIONS

[0229] In some aspects, the compositions disclosed herein (e.g, polynucleotides encoding CARs of the present disclosure, vectors comprising polynucleotides encoding CARs of the present disclosure, CARs of the present disclosure, or cells expressing CARs of the present disclosure, .eg., CAR-T cells) can be used to treat a disease or condition, e.g, a proliferative disease such as a cancer or malignancy or a precancerous condition such as a myelodysplasia, a myelodysplastic syndrome or a preleukemia.

[0230] A "cancer" refers to a broad group of various proliferative diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth results in the formation of malignant tumors that invade neighboring tissues and can also metastasize to distant parts of the body through the lymphatic system or bloodstream. As used herein the term "proliferative" disorder or disease refers to unwanted cell proliferation of one or more subset of cells in a multicellular organism resulting in harm (i.e., discomfort or decreased life expectancy) to the multicellular organism. For example, as used herein, proliferative disorder or disease includes neoplastic disorders and other proliferative disorders. "Neoplastic," as used herein, refers to any form of dysregulated or unregulated cell growth, whether malignant or benign, resulting in abnormal tissue growth. Thus, "neoplastic cells" include malignant and benign cells having dysregulated or unregulated cell growth. In some aspects, the cancer is a tumor. "Tumor," as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.

[0231] In some aspects, the disease is a solid or a liquid tumor. In some aspects, the cancer is a pancreatic cancer. In some aspects, the disease is a hematologic cancer. In some aspects, the hematologic cancer is a leukemia. In some aspects, the cancer is selected from the group consisting of one or more acute leukemias including but not limited to B-cell acute lymphoid leukemia (BALL), T-cell acute lymphoid leukemia (TALL), small lymphocytic leukemia (SLL), acute lymphoid leukemia (ALL) ( e g ., relapsing and refractory ALL); one or more chronicPCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1 leukemias including but not limited to chronic myelogenous leukemia (CML), and chronic lymphocytic leukemia (CLL). Additional hematologic cancers or conditions include, but are not limited to mantle cell lymphoma (MCL), B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitf s lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell- or a large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, Marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin lymphoma, Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, and preleukemia. Preleukemia encompasses a diverse collection of hematological conditions united by ineffective production (or dysplasia) of myeloid blood cells. In some aspects, the indication is an atypical and / or non-classical cancer, malignancy, precancerous condition or proliferative disease; and any combination thereof

[0232] In some aspects, the disease is a lymphoma, e.g., MCL or Hodgkin lymphoma. In some aspects, the disease is leukemia, e.g, SLL, CLL and / or ALL. In some aspects, the disease associated with a tumor antigen, e.g., a tumor antigen described herein, is selected from a proliferative disease such as a cancer or malignancy or a precancerous condition such as a myelodysplasia, a myelodysplastic syndrome or a preleukemia, or is a non-cancer related indication associated with expression of a tumor antigen described herein. In some aspects, the disease associated with a tumor antigen described herein is a solid tumor, e.g, a solid tumor described herein, e.g, prostatic, colorectal, pancreatic, cervical, gastric, ovarian, head, or lung cancer.

[0233] In some aspects, the cancer is chosen from AML, ALL, B-ALL, T-ALL, B-cell prolymphocytic leukemia, chronic lymphocytic leukemia, CML, hairy cell leukemia, Hodgkin lymphoma, mast cell disorder, myelodysplastic syndrome, myeloproliferative neoplasm, plasma cell myeloma, plasmacytoid dendritic cell neoplasm, or a combination thereof.

[0234] In some aspects, the compositions disclosed herein (e.g, polynucleotides encoding CARs of the present disclosure, vectors comprising polynucleotides encoding CARs of the present disclosure, CARs of the present disclosure, or cells expressing CARs of the present disclosure, e.g., CAR-T cells) are used to reduce or decrease a size of a tumor or inhibit a tumor growth in a subject in need thereof. In some aspects, the tumor is a carcinoma (i.e., a cancer of epithelial origin). In some aspects, the tumor is, e.g., selected from the group consisting ofPCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1 gastric cancer, gastroesophageal junction cancer (GEJ), esophageal cancer, colorectal cancer, liver cancer (hepatocellular carcinoma, HCC), ovarian cancer, breast cancer, NSCLC, bladder cancer, lung cancer, pancreatic cancer, head and neck cancer, lymphoma, uterine cancer, renal or kidney cancer, biliary cancer, prostate cancer, testicular cancer, urethral cancer, penile cancer, thoracic cancer, rectal cancer, brain cancer (glioma and glioblastoma), cervical cancer, parotid cancer, larynx cancer, thyroid cancer, adenocarcinomas, neuroblastomas, melanoma, and Merkel Cell carcinoma.

[0235] A "cancer" or "cancer tissue" can include a tumor at various stages. In certain aspects, the cancer or tumor is stage 0, such that, e.g., the cancer or tumor is very early in development and has not metastasized. In some aspects, the cancer or tumor is stage I, such that, e.g., the cancer or tumor is relatively small in size, has not spread into nearby tissue, and has not metastasized. In other aspects, the cancer or tumor is stage II or stage III, such that, e.g., the cancer or tumor is larger than in stage 0 or stage I, and it has grown into neighboring tissues, but it has not metastasized, except potentially to the lymph nodes. In other aspects, the cancer or tumor is stage IV, such that, e.g., the cancer or tumor has metastasized. Stage IV can also be referred to as advanced or metastatic cancer.

[0664] In some aspects, the cancer can include, but is not limited to, adrenal cortical cancer, advanced cancer, anal cancer, aplastic anemia, bileduct cancer, bladder cancer, bone cancer, bone metastasis, brain tumors, brain cancer, breast cancer, childhood cancer, cancer of unknown primary origin, Castleman disease, cervical cancer, colon / rectal cancer, endometrial cancer, esophagus cancer, Ewing family of tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gestational trophoblastic disease, Hodgkin disease, Kaposi sarcoma, renal cell carcinoma, laryngeal and hypopharyngeal cancer, acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myelomonocytic leukemia, liver cancer, non-small cell lung cancer, small cell lung cancer, lung carcinoid tumor, lymphoma of the skin, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumors, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma in adult soft tissue, basal and squamous cell skin cancer, melanoma, small intestine cancer, stomach cancer, testicular cancer, throat cancer, thymus cancer, thyroid cancer,PCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1 uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, Wilms tumor and secondary cancers caused by cancer treatment.

[0236] In some aspects, the tumor is a solid tumor. A "solid tumor" includes, but is not limited to, sarcoma, melanoma, carcinoma, or other solid tumor cancer. "Sarcoma" refers to a tumor which is made up of a substance like the embryonic connective tissue and is generally composed of closely packed cells embedded in a fibrillar or homogeneous substance. Sarcomas include, but are not limited to, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abernethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, immunoblastic sarcoma of B cells, lymphoma, immunoblastic sarcoma of T-cells, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymoma sarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma, or telangiectaltic sarcoma.

[0237] The term "melanoma" refers to a tumor arising from the melanocytic system of the skin and other organs. Melanomas include, for example, acra-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, metastatic melanoma, nodular melanoma, subungal melanoma, or superficial spreading melanoma.

[0238] The term "carcinoma" refers to a malignant new growth made up of epithelial cells tending to infiltrate the surrounding tissues and give rise to metastases. Exemplary carcinomas include, e.g., acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchi oalveol ar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiermoidPCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1 carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum, gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma, carcinoma gigantocellulare, glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypemephroid carcinoma, infantile embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher's carcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullary carcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum, carcinoma mucocellulare, mucoepidemoid carcinoma, carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes, naspharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reserve cell carcinoma, carcinoma sarcomatodes, Schneiderian carcinoma, scirrhous carcinoma, carcinoma scroti, signetring cell carcinoma, carcinoma simplex, small-cell carcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamous carcinoma, squamous cell carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, or carcinoma viflosum.

[0239] Additional cancers that can be treated with the compositions disclosed herein (e.g., polynucleotides encoding CARs of the present disclosure, vectors comprising polynucleotides encoding CARs of the present disclosure, CARs of the present disclosure, or cells expressing CARs of the present disclosure, e.g., CART cells) include, e.g., Leukemia, Hodgkin's Disease, Non- Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, smallcell lung tumors, primary brain tumors, stomach cancer, colon cancer, malignant pancreatic insulanoma, malignant carcinoid, urinary bladder cancer, premalignant skin lesions, testicular cancer, lymphomas, thyroid cancer, papillary thyroid cancer, neuroblastoma, neuroendocrine cancer, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, adrenal cortical cancer, prostate cancer, Miillerian cancer, ovarian cancer, peritoneal cancer, fallopian tube cancer, or uterine papillary serous carcinoma.PCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1

[0240] METHODS

[0241] The present disclosure also provide methods for using of the CARs and CAR-expressing cells of the present disclosure for adoptive therapy. In some aspects, the present disclosure provides a method of stimulating a T cell-mediated immune response to a target cell population or tissue in a subject, comprising administering an effective amount of a cell expressing a CAR of the present disclosure to the subject. Also provided is a method of providing an anti-tumor immunity in a subject in need thereof, the method comprising administering to the subject an effective amount of a cell expressing a CAR of the present disclosure to the subject.

[0242] The disclosure also provides a method of treating cancer in a subject in need thereof comprising administering to the subject an effective amount of a cell expressing a CAR of the present disclosure. The disclosure also provides a method of preparing a population of cells, e.g. CART cells, for a therapy comprising transducing a population of cells isolated from a subject with a polynucleotide or vector of the present disclosure. In some aspects, the transduction comprises culturing the cell under suitable condition.

[0243] The disclosure also provides a method of generating a persisting population of genetically engineered cells in a subject diagnosed with cancer, the method comprising administering to the subject a cell genetically engineered to express a CAR of the present disclosure.

[0244] The disclosure also provides a method of expanding a population of genetically engineered cells (e.g., T cells) in a subject diagnosed with cancer, the method comprising administering to the subject a cell (e.g, a T cell) genetically engineered to express a CAR of the present disclosure. In some aspects, the cell is a T cell, e.g, an autologous T cell. In other aspects, the T cell is a heterologous T cell. In some aspects of the methods disclosed herein, the subject is a human subject.

[0245] The present disclosure also provides a method to improve one or more properties of a CAR therapy comprising inserting a CAR spacer of the present disclosure between an antigenbinding domain and a transmembrane domain of a CAR, wherein the spacer is located between the ligand-binding domain and the transmembrane domain.

[0246] In some aspects, the one or more improved properties of the CAR therapy is increased secretion of one or more cytokines. In some aspects, the cytokine secretion induced by the CARPCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1 of the present disclosure, i.e., a CAR comprising a CAR spacer of the present disclosure, is increased with respect to the secretion observed after administration of a corresponding CAR comprising no spacer. In some aspects, the cytokine is an interferon, e.g, interferon-gamma. In some aspects, the cytokine is a tumor necrosis factor (TNF), e.g, TNF-alpha.

[0247] In some aspects, administration of a CAR of the present disclosure results in an increase in interferon (e.g, interferon-gamma) secretion by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% compared to the interferon (e.g, interferon- gamma) secretion observed after administration of a corresponding CAR comprising no spacer.

[0248] In some aspects, administration of a CAR of the present disclosure results in an increase in TNF-alpha secretion by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% compared to the TNF-alpha secretion observed after administration of a corresponding CAR comprising no spacer.

[0249] In some aspects, the present disclosure provides a method to select an optimal CAR spacer comprising comparing killing kinetics (Area Under Curve) versus cytokine (e.g., IFN- gamma and / or TNF-alpha) in a library of CARs differing in the spacer used (e.g., the spacer is a spacer from the library provided in TABLE 2), and selecting the CAR with the lowest AUC and / or highest cytokine. Also provided is a method to optimize or improve a CAR comprising replacing the CAR’s original spacer with a spacer disclosed herein (e.g., a spacer from the library provided in TABLE 2), comparing killing kinetics (Area Under Curve) and cytokine production (e.g., IFN-gamma and / or TNF-alpha), and selecting the CAR with the lowest AUC and / or highest cytokine production.

[0250] The present disclosure also provides a method to select an optimal spacer comprising generating a library of CARs differing in the spacer used (e.g., the spacer is a spacer from the library provided in TABLE 2 or any spacer disclosed herein) and performing sequential kill experiments in which CAR-T cells are continually passaged into new target cells until the CAR-PCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1T cells stop proliferating and killing. The best CAR spacers would be those present in the CAR- T cells that would have a more prolonged proliferation and killing activity.

[0251] In some aspects, the present disclosure provides a polynucleotide, vector, CAR, composition, kit, cell, or the pharmaceutical composition of the present disclosure for use as a medicament. In some aspects, the present disclosure provides a polynucleotide, vector, CAR, composition, kit, cell, or the pharmaceutical composition of the present disclosure for use as a medicament for the treatment of cancer in a subject in need thereof. In some aspects, the present disclosure provides a polynucleotide, vector, CAR, composition, kit, cell, or the pharmaceutical composition of the present disclosure for the treatment of cancer in a subject in need thereof. In some aspects, the present disclosure provides the use of a polynucleotide, vector, CAR, composition, kit, cell, or the pharmaceutical composition of the present disclosure for the manufacture of a medicament. In some aspects, the present disclosure provides the use of a polynucleotide, vector, CAR, composition, kit, cell, or the pharmaceutical composition of the present disclosure for the manufacture of a medicament for treating cancer in a subject in need thereof.

[0252] The present disclosure also provides a composition comprising a polynucleotide encoding a CAR, a vector comprising a polynucleotide encoding a CAR, or a genetically modified cell comprising a polynucleotide or a vector encoding a CAR for treating a subject in need of a CAR therapy. The present disclosure also provides a composition comprising a polynucleotide encoding a CAR, a vector comprising a polynucleotide encoding a CAR, or a genetically modified cell comprising a polynucleotide or a vector encoding a CAR for use as a medicament. Also provided is a composition comprising a polynucleotide encoding a CAR, a vector comprising a polynucleotide encoding a CAR, or a genetically modified cell comprising a polynucleotide or a vector encoding a CAR for used as treatment for cancer in a subject in need of a CAR therapy. Also provided is a composition comprising a polynucleotide encoding a CAR, a vector comprising a polynucleotide encoding a CAR, or a genetically modified cell comprising a polynucleotide or a vector encoding a CAR for the manufacture of a medicament for the treatment for cancer in a subject in need of a CAR therapy.

[0253] KITSPCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1

[0254] The present disclosure also provides kits, or products of manufacture comprising (i) a CAR of the present disclosure (i.e., CAR comprising a CAR spacer of the present disclosure, i.e., a human IgGl hinge and a human IgGl Fc derived CAR spacer), one or more polynucleotides encoding a CAR of the present disclosure, one or more vectors encoding a CAR of the present disclosure, or a composition comprising the polynucleotide(s) or vector(s), and optionally (ii) instructions for use, e.g., instructions for use according to the methods disclosed herein.

[0255] The disclosure also provides a kits comprising (i) a cell genetically modified to express a CAR of the present disclosure, i.e., a cell one or more polynucleotides encoding a CAR of the present disclosure, or one or more vectors encoding a CAR of the present disclosure (e.g, a T cell, a natural killer (NK) cell, an natural killer T (NKT) cell, or an ILC cell), or a pharmaceutical composition comprising the cell, and optionally (ii) instructions for use.

[0256] In some aspects, the kit or product of manufacture comprises at least a polynucleotide or vector encoding a CAR of the present disclosure, a cell genetically modified to express a CAR of the present disclosure, or a composition (e.g, a pharmaceutical composition) comprising a polynucleotide, vector, or cell disclosed herein, in one or more containers.

[0257] In some aspects, the kit or product of manufacture comprises at least a polynucleotide or vector encoding a CAR of the present disclosure, a cell genetically modified to express a CAR of the present disclosure, or a composition (e.g., a pharmaceutical composition) comprising a polynucleotide, vector, or cell disclosed herein, and optionally a brochure.

[0258] One skilled in the art will readily recognize that the polynucleotides, vectors, cells, and compositions of the present disclosure, pharmaceutical composition comprising the polynucleotides, vectors, or cells of the present disclosure, or combinations thereof can be readily incorporated into one of the established kit formats which are well known in the art.

[0259] In some aspects, the kit or product of manufacture comprises, e.g., a polynucleotide or vector encoding a CAR of the present disclosure, or a composition (e.g, a pharmaceutical composition) comprising a polynucleotide, vector, in dry form in a container (e.g, a glass vial), and optionally a vial with a solvent.

[0260] In some aspects, the kit or product of manufacture comprises, e.g, a polynucleotide or vector encoding a CAR of the present disclosure, or a composition (e.g, a pharmaceutical composition) comprising a polynucleotide, vector, in at least one container, and another or more containers with transfection reagents.PCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1

[0261] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Sambrook et ah, ed. (1989) Molecular Cloning A Laboratory Manual (2nd ed.; Cold Spring Harbor Laboratory Press); Sambrook et ah, ed. (1992) Molecular Cloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); D. N. Glover ed., (1985) DNA Cloning, Volumes I and II; Gait, ed. (1984) Oligonucleotide Synthesis; Mullis et al. U.S. Pat. No. 4,683,195; Hames and Higgins, eds. (1984) Nucleic Acid Hybridization; Hames and Higgins, eds. (1984) Transcription And Translation; Freshney (1987) Culture Of Animal Cells (Alan R. Liss, Inc.); Immobilized Cells And Enzymes (IRL Press) (1986); Perbal (1984) A Practical Guide To Molecular Cloning; the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Miller and Calos eds. (1987) Gene Transfer Vectors For Mammalian Cells, (Cold Spring Harbor Laboratory); Wu et al., eds., Methods In Enzymology, Vols. 154 and 155; Mayer and Walker, eds. (1987) Immunochemical Methods In Cell And Molecular Biology (Academic Press, London); Weir and Blackwell, eds., (1986) Handbook Of Experimental Immunology, Volumes I-IV; Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1986); ); Crooke, Antisense drug Technology: Principles, Strategies and Applications, 2nd Ed. CRC Press (2007) and in Ausubel et al. (1989) Current Protocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.).

[0262] ENUMERATED EMBODIMENTS

[0263] A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the following examples are intended to illustrate but not limit the scope of invention described in the claims

[0264] Embodiment 1 is an chimeric antigen receptor (CAR) comprising(i) an extracellular binding domain that binds to a tumor-associated antigen;(ii) a transmembrane domain;PCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1(iii) an intracellular domain; and,(iv) a spacer located between the extracellular binding domain and the transmembrane domain comprising an amino acid sequence derived from a hinge region and a crystallizable fragment (Fc) of a human immunoglobulin G1 isotype (IgGl).

[0265] Embodiment 2 is the CAR of embodiment 1, wherein the CAR is designed as a second- generation CAR.

[0266] Embodiment 3 is the CAR of embodiment 1 or 2, wherein the transmembrane domain comprises a CD8a transmembrane region (CD8a-TM) polypeptide.

[0267] Embodiment 4 is the CAR of embodiment 3, wherein the CD8a-TM polypeptide comprises an amino acid sequence of SEQ ID NO: 8.

[0268] Embodiment 5 is the CAR of any one of embodiments 1 to 4, wherein the intracellular domain comprises a TNF receptor superfamily member 9 (CD137) component and / or a T-cell surface glycoprotein CD3 zeta chain (CD3z) component.

[0269] Embodiment 6 is the CAR of embodiment 5, wherein the CD137 component comprises an amino acid sequence of SEQ ID NO: 9.

[0270] Embodiment 7 is the CAR of embodiment 5, wherein the CD3z component comprises an amino acid sequence of SEQ ID NO: 10.

[0271] Embodiment 8 is the CAR of any one of embodiments 1 to 7, wherein the spacer comprises an amino acid sequence derived from the hinge, CH2, and CH3 regions of the human IgGl.

[0272] Embodiment 9 is the CAR of any one of embodiments 1 to 8, wherein the spacer comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:6.

[0273] Embodiment 10 is a polynucleotide encoding the CAR of any one of embodiments 1 to 9.

[0274] Embodiment 11 is a vector comprising the polynucleotide of embodiment 10.

[0275] Embodiment 12 is the vector of embodiment 11, wherein the vector is a lentiviral vector.

[0276] Embodiment 13 is a cell genetically modified to express the CAR of any one of embodiments 1 to 9.

[0277] Embodiment 14 is the cell of embodiment 13, wherein the cell is a T cell.

[0278] Embodiment 15 is a composition comprising the CAR of any one of embodiments 1 to 9.PCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1

[0279] Embodiment 16 is a pharmaceutical composition comprising the cell of embodiment 13 or the composition of embodiment 15.

[0280] Embodiment 17 is a method to improve one or more properties of a CAR comprising inserting a CAR spacer between the extracellular binding domain and the transmembrane domain comprising an amino acid sequence derived from a hinge region and a crystallizable fragment (Fc) of a human immunoglobulin G1 isotype (IgGl).

[0281] Embodiment 18 is the method of embodiment 17, wherein the spacer comprises an amino acid sequence derived from the hinge, CH2, and CH3 regions of the human IgGl .

[0282] Embodiment 19 is the method of embodiment 17 or 18, wherein the spacer comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 6.

[0283] Embodiment 20 is the method of any one of embodiments 17 to 19, wherein the one or more improved properties is increased secretion of one or more cytokines or increased tumor cell conjugation.

[0284] Embodiment 21 is the method of any one of embodiments 17 to 20, wherein the cytokine is an interferon.

[0285] Embodiment 22 is the method of embodiment 21, wherein the interferon is interferongamma.

[0286] Embodiment 23 is the method of any of embodiments 17 to 20, wherein the cytokine is a tumor necrosis factor.

[0287] Embodiment 24 is method of embodiment 23, wherein the tumor necrosis factor is tumor necrosis factor-alpha.

[0288] Embodiment 25 is a method of treating cancer in a subject in need thereof comprising administering to the subject an effective amount of the cell of embodiment 13.

[0289] Embodiment 26 is a method of preparing a population of cells for a therapy comprising transducing a population of cells isolated from a subject with the polynucleotide of embodiment 10 or the vector of embodiment 11.

[0290] Embodiment 27 is a chimeric antigen receptor (CAR) comprising:(iv) an extracellular binding domain that binds to a tumor-associated antigen;(v) a transmembrane domain; and(vi) an intracellular domain, the improvement comprising a spacer comprising SEQ ID NO: 6.PCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1EXAMPLESEXAMPLE 1. UNRAVELING THE IMPACT OF CAR AFFINITY AND SPACER LENGTH ON T CELL FUNCTION: DESIGN, CONSTRUCTION, AND EVALUATION OF MODIFIED ANTI-DLL3 AND ANTI-BCMA CARS

[0291] To comprehensively understand the influence of CAR affinity and spacer length on T cell function, we meticulously designed and tested CARs targeting DLL3 and BCMA. We utilized DLL3-specific and BCMA-specific scFv L-H orientation domains, which were derived from parental antibodies DL3B469 and BCMB519, respectively.Table 1. Heavy Chain and Light Chain Sequences of Parental Antibodies DL3B469 andBCMB519PCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1

[0292] To manipulate antigen binding strength, amino acid substitutions were introduced in the CDR of each scFv (FIG. 1A).

[0293] The affinity variant scFv constructs, showcasing the desired binding characteristics, were paired with a short, intermediate, or long spacer for CAR design: CD8 alpha, IgGl hinge-CH2- CH3, or tandem IgGl hinge-CH2-CH3-IgG4hinge-CH2-CH3, respectively. The IgGl and IgG4PCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1 hinge sequences used are EPKSSDKTHTCPPCP (SEQ ID NO: 40) and ESKYGPPCPPCP (SEQ ID NO: 41) and they are shown in parenthesis in Table 2.

[0294] All CAR constructs adhered to the second generation design , featuring a CD8 transmembrane domain, 4-1BB costimulatory domain, and CD3(^ signaling domain.Table 2. Amino Acid Sequences of Spacers Used for CAR Design:Table 3. Amino Acid Sequences of Second Generation CAR DomainsPCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1

[0295] CAR-encoded transgenes were efficiently cloned into a pLLV lentiviral vector.Lentivirus production was executed using the LV Max suspension cell system kit (Thermofisher), coupled with pseudoviral packaging plasmids. This intricate approach, outlined in more detail in the following sections, allowed us to systematically evaluate the designed affinity and spacer variant CARs for their impact on effector function (FIG. IB).EXAMPLE 2. COMPREHENSIVE EVALUATION OF ANTI-DLL3 AND ANTLBCMA SCFV-FC PROTEINS: AFFINITY PROFILING AND SINGLE ARM ANTIGEN BINDING ANALYSIS

[0296] We conducted a thorough examination of anti-DLL3 and anti-BCMA scFv-Fc proteins, exploring affinity variants created through CDR sequence manipulation of parental DLL3 and BCMA antibodies. These variants, featuring amino acid mutations in the CDR region, were expressed and evaluated for their binding to DLL3+ or BCMA+ tumor cells and recombinant proteins.

[0297] For anti-DLL3 scFvs, including DLL3.A, DLL3.B, DLL3.C, and DLL3.D, descending order of binding strength was identified. Flow cytometry cell binding analysis on DLL3+ SHP- 77 cells unveiled a substantial 42-fold range of affinities (mean EC50: DLL3.A = 12.5nM, DLL3.B = 42.2nM, DLL3.C = 114.7nM, DLL3.D = 525.6nM) (FIG. 2A). Additionally, these scFvs underwent screening for binding to recombinant DLL3 protein, with SPR analysisPCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1 revealing a nearly 1000-fold range of affinities (mean KD: DLL3.A = 0.41nM, DLL3.B = 7.2nM, DLL3.C = 47nM, DLL3.D = 407nM) (FIG.2D).Table 4. DLL3 ScFv Amino Acid Sequences

[0298] Similarly, for anti-BCMA scFvs, including BCMA.A, BCMA.B, and BCMA.C, descending order of binding strength was identified. Flow cytometry cell binding analysis on BCMA+ H929 cells demonstrated a significant 36-fold range of affinities (mean EC50: BCMA.A = 3.6nM, BCMA.B = 30.9nM, BCMA.C = 131.7nM) (FIG. 2C). Additionally, these scFvs were screened for binding to recombinant BCMA protein, with SPR analysis revealing aPCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1414-fold range of affinities (mean KD: BCMA.A = 0.18nM, BCMA.B = 17nM, BCMA.C = 74.6nM) (FIG. 2D).Table 5. BCMA ScFv Amino Acid SequencesEXAMPLE 3. DESIGN AND CHARACTERIZATION OF AFFINITY VARIANT AND SPACER VARIANT BCMA AND DLL3 CARS

[0299] We engineered affinity variant scFvs, as detailed earlier, into L-H orientation CARs with varied-length spacer domains. Each scFv was paired with one of three spacer domains: CD8a (short), IgGl CH2-CH3 (intermediate), or tandem IgGl (CH2-CH3)2 (long). All CARs adhered to the second-generation model, encompassing 4-1BB and CD3(^ signaling domains, along with a CD8 transmembrane domain (FIG. 3A and FIG. 3B).Table 6. Amino Acid Sequences of DLL3 and BCMA CARs Comprising Short, Intermediate, and Long SpacersPCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1PCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1PCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1PCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1PCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1PCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1PCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1PCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1PCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1PCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1

[0300] Synthesis and cloning of CAR transgenes facilitated their packaging into lentiviral particles. Subsequently, purified T cells from healthy donors underwent transduction with CAR- encoded lentiviral particles. Transduction efficiency and CAR surface expression were validated using a proprietary anti-CAR monoclonal antibody.PCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1

[0301] DLL3 CARs exhibited a transduction efficiency range of 28.2% to 59.9%, accompanied by relatively similar surface expression levels (FIG. 3C). Notably, DLL3 CARs with long spacers displayed slightly lower surface expression compared to their short or intermediate spacer counterparts. On the other hand, BCMA CARs demonstrated a transduction efficiency range of 40% to 68.6%, with surface expression levels that were relatively similar (FIG. 3D). Interestingly, BCMA CARs with intermediate-length spacers exhibited slightly higher surface expression compared to short or long spacer CARs.

[0302] To ensure standardized comparisons, all CAR T cell populations underwent normalization based on %CAR+ using control T cells before proceeding to functional evaluations. This comprehensive characterization provides a foundation for understanding the nuances in surface expression and transduction efficiency among the designed affinity variant and spacer variant BCMA and DLL3 CARs.EXAMPLE 4. EXPLORATION OF CAR BIOPHYSICAL PROPERTIES: CAR CLUSTERING AND CELLULAR AVIDITY MEASUREMENTS

[0303] In our pursuit of understanding the intricate dynamics of CAR biophysical properties, we embarked on a multifaceted exploration, encompassing receptor clustering and cellular avidity measurements.

[0304] Receptor Clustering Analysis:

[0305] Recombinant human DLL3 and BCMA were directly biotinylated at a 1 : 1 biotin: antigen ratio. Subsequently, biotinylated antigens were conjugated to streptavidin-Alexa Fluor 647 (Jackson ImmunoResearch) at a 3 : 1 ratio. CAR T cells were then exposed to dilutions of the antigen complexes for 1 hour and analyzed on an Intellicyt iQue 3 flow cytometer. High affinity CARs yielded tighter antigen complex binding compared to lower affinity CARs, suggesting a consistent trend and relationship between single antigen CAR engagement (affinity) and CAR clustering (avidity) (FIG 4A and FIG. 4B). The outcome of this analysis provided insights into affinity-dependent antigen binding potency in the context of CAR clustering avidity.Interestingly, the results revealed similar binding potency across spacer domains, implying a lack of spacer-dependent clustering advantages.

[0306] Cellular Avidity Measurements-Conjugation Assay:PCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1

[0307] For the cellular avidity measurements, CAR T cells were stained with 0.05pM Cell Tracker Deep Red (CTDR; Invitrogen), while tumor cells were stained with 0.5pM Cell Trace Violet (CTV; Invitrogen). Effector and tumor cells were then plated in 96-well round-bottom plates at a 1 : 1 effector to tumor ratio (E:T). Co-cultures underwent centrifugation for 3 minutes at 125 x g and were subsequently incubated for 10 minutes. The analysis on an Intellicyt iQue 3 flow cytometer enabled the definition of cell conjugates, with effector cells gated as CTV+ / CTDR+. Analysis revealed minimal affinity-dependent effects on cellular avidity, however CARs paired with CH2-CH3 spacers yielded the highest percentage of tumor cell conjugates, proposing structural advantages for tumor cell engagement (FIG. 4C and FIG. 4D).EXAMPLE 5. COMPREHENSIVE ANALYSIS OF CAR SIGNALING DYNAMICS

[0308] In our exploration of CAR signaling, we employed a dual reporter system to measure the output in response to both tumor cells and soluble antigen complexes. Initially, a reporter Jurkat cell line was obtained from Systems Biosciences, integrating an NF-kB / GFP reporter system and later modified to include an mCherry / Nur77 reporter system. Subsequently, these dual reporter Jurkat cells, each equipped with specific CAR designs, underwent a 24-hour coincubation with tumor cells.

[0309] The subsequent flow cytometry analysis, conducted on a BD Symphony instrument, enabled the assessment of CAR+ effector cells for GFP (NF-kB) and mCherry (Nur77) dual expression.

[0310] The results unveiled that CAR signaling tends to decrease for DLL3 -specific CARs when affinity is weaker than ~1 OnM KD (FIG. 4E). Interestingly, affinity variant BCMA- specific CARs elicited comparable signaling strength, suggesting a more sensitive affinity threshold for optimal signaling (FIG. 4F). Additionally, responses to tumor cells exhibited maximal strength for short spacer CARs, with potency diminishing as spacer length increased.

[0311] This comprehensive analysis, incorporating the additional insights from the stable transduction of DLL3 -specific and BCMA-specific CARs, enhances our understanding of the intricate relationship between CAR affinity, spacer length, and the resulting T-cell signaling dynamics.PCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1EXAMPLE 6: PROLONGED EFFECTOR FUNCTION OF AFFINITY AND SPACER VARIANT DLLS AND BCMA CAR T CELLS

[0312] In our pursuit to grasp the enduring impact of CAR affinity and spacer length on effector function, we conducted a serial stimulation spheroid assay, systematically evaluating each CAR panel. GFP-expressing tumor cells were deliberately seeded and cultured in ultra-low attachment plates for 3 days, fostering the formation of spheroids. DLL3 or BCMA CARs were subsequently introduced at varying E:T ratios and co-cultured for a span of 10 days. The trajectory of tumor growth and fluorescent intensity was meticulously monitored using the Incucyte SX5 instrument. Endpoint flow cytometry analyses were then executed to quantify the proliferation of CAR+ T cells. Additionally, supernatants were harvested to quantify proinflammatory cytokines using MSD V plex kits.

[0313] DLL3 CARs, specifically those paired with intermediate-length (IgGl CH2-CH3) spacers, exhibited equivalent or superior tumor lysis and T cell expansion compared to their counterparts with short or long spacers (FIG. 5A). These particular CARs also demonstrated heightened secretion of proinflammatory cytokines, including IFN-y and TNF-a (FIG. 5B). Intriguingly, the DLL3 CAR with the lowest affinity (DLL3.D) showcased suboptimal tumor lysis and proinflammatory cytokine production, yet it demonstrated comparable T cell expansion to higher affinity CARs.

[0314] Turning our attention to the BCMA CAR panel, we observed substantial functional advantages for CARs paired with intermediate-length (IgGl CH2-CH3) spacers. Especially under medium to low E:T conditions, these CARs effectively dismantled tumor spheroids and instigated robust T cell expansion (FIG. 6A). These findings were corroborated by the quantified cytokine secretion (FIG. 6B). Interestingly, minimal functional differences were discerned for the affinity variant CARs, hinting at a decline in function only when CAR affinity is weaker than lOOnM (KD).

Claims

PCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT1We claim:

1. A chimeric antigen receptor (CAR) comprising:(i) an extracellular binding domain that binds to a tumor-associated antigen;(ii) a transmembrane domain;(iii) an intracellular domain; and,(iv) a spacer located between the extracellular binding domain and the transmembrane domain, the spacer comprising an amino acid sequence derived from a hinge region and a crystallizable fragment (Fc) of a human immunoglobulin G1 isotype (IgGl).

2. The CAR of claim 1, wherein the CAR is designed as a second-generation CAR.

3. The CAR of claim 1 or 2, wherein the transmembrane domain comprises a CD8a transmembrane region (CD8a-TM) polypeptide.

4. The CAR of claim 3, wherein the CD8a-TM polypeptide comprises SEQ ID NO: 8.

5. The CAR of any one of claims 1 to 4, wherein the intracellular domain comprises a TNF receptor superfamily member 9 (CD 137) component and / or a T-cell surface glycoprotein CD3 zeta chain (CD3z) component.

6. The CAR of claim 5, wherein the CD137 component comprises the amino acid sequence of SEQ ID NO: 9.

7. The CAR of claim 5, wherein the CD3z component comprises the amino acid sequence of SEQ ID NO: 10.

8. The CAR of any one of claims 1 to 7, wherein the spacer comprises an amino acid sequence derived from the hinge, CH2, and CH3 regions of human IgGl.

9. The CAR of any one of claims 1 to 8, wherein the spacer comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 6.

10. A polynucleotide encoding the CAR of any one of claims 1 to 9.

11. A vector comprising the polynucleotide of claim 10.

12. The vector of claim 11, wherein the vector is a lentiviral vector.

13. A cell genetically modified to express the CAR of any one of claims 1 to 9.

14. The cell of claim 13, wherein the cell is a T cell.

15. A composition comprising the CAR of any one of claims 1 to 9.PCT / US25 / 31343 29 May 2025 (29.05.2025)Attorney Docket No.: JBI6913WOPCT116. A pharmaceutical composition comprising the cell of claim 13 or the composition of claim 15.

17. A method for improving one or more properties of a CAR, the method comprising inserting a CAR spacer between the extracellular binding domain and the transmembrane domain, wherein the spacer comprises an amino acid sequence derived from a hinge region and a crystallizable fragment (Fc) of a human immunoglobulin G1 isotype (IgGl ).

18. The method of claim 17, wherein the spacer comprises an amino acid sequence derived from the hinge, CH2, and CH3 regions of human IgGl.

19. The method of claim 17 or 18, wherein the spacer comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:

620. The method of any one of claims 17 to 19, wherein the one or more improved properties include increased secretion of one or more cytokines or increased tumor cell conjugation.

21. The method of any one of claims 17 to 20, wherein the cytokine is an interferon.

22. The method of claim 21, wherein the interferon is interferon-gamma.

23. The method of any one of claims 17 to 20, wherein the cytokine is a tumor necrosis factor.

24. The method of claim 23, wherein the tumor necrosis factor is tumor necrosis factor-alpha.

25. A method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of the cell of claim 13.

26. A method of preparing a population of cells for a therapy, the method comprising transducing a population of cells isolated from a subject with the polynucleotide of claim 10 or the vector of claim 11.

27. A CAR comprising:(i) an extracellular binding domain that binds to a tumor-associated antigen;(ii) a transmembrane domain; and(iii) an intracellular domain, wherein the CAR comprises an improvement featuring a spacer that comprises the amino acid sequence of SEQ ID NO: 6.

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