Polypeptides that bind to CD5 and uses thereof

Abstract

Embodiments provided herein, provide for polypeptides, pharmaceutical compositions, and methods that can be used, for example, to treat cancers or other immune cell mediated diseases, such as those that bind to CDS.

Description

[0001] DOCKET NO. VTB-013WO PATENT POLYPEPTIDES THAT BIND TO CD5 AND USES THEREOF

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit of U. S. Provisional Application Ser. No.

[0004] 63 / 753,611 filed February 04, 2025, which is hereby incorporated by reference in its entirety.

[0005] REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0006] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on February 03, 2026, is named “VTB-013WO_SL.xml” and is 25,815 bytes in size.

[0007] FIELD

[0008] The embodiments provided herein relate to compositions that target different cells to regulate an immune response, such as for the treatment of cancer, autoimmunity, and / or infectious diseases.

[0009] BACKGROUND

[0010] Cell-mediated immunity plays a critical role in the body’s immune response to many diseases, including cancer, autoimmunity, or infectious diseases. Dysregulation of immune cells can also lead to susceptibility to pathogens and autoimmune disease. Unfortunately, in many disease states, therapeutics lose their efficacy due to the immune system turning itself off or otherwise becoming less active to attack and kill pathogenic cells. Thus, there is a need for therapeutics that can activate or re-activate the immune system or down-regulate aberrant immune cell function to treat cancer and other diseases. The embodiments provided herein fulfill these needs as well as others.

[0011] SUMMARY

[0012] In some embodiments, a polypeptide is provided, the polypeptide comprising a first binding domain (effector moiety) and a second binding domain (effector moiety), wherein the first binding domain binds to an extracellular domain of CD5; and the second binding domain binds to an immune cell surface protein. In some embodiments, the second binding domain binds to an extracellular domain of CD5. In some embodiments, the first binding domain and

[0013] -1- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT

[0014] the second binding domain bind to different CD5 epitopes. In some embodiments, the first binding domain and the second binding domain bind to the same CD5 epitope.

[0015] In some embodiments, the polypeptide comprises a third binding domain (effector moiety) that binds to a cell specific antigen, such as a target cell or tumor antigen, or an immune cell surface antigen that is a different protein from what the first binding domain and the second binding domain do. In some embodiments, the target cell antigen is CD 19, CD22, TAG-72, MUC16, PSMA, EGFR, a-integrin, BCM A, HER2, or Mesothelin, CLDN6, Nectin-4, CEA, and the like.

[0016] In some embodiments, a pharmaceutical composition is provided. In some embodiments, the pharmaceutical composition comprises a polypeptide as provided for herein.

[0017] In some embodiments, a nucleic acid molecule or molecules is provided. In some embodiments, the nucleic acid molecule or molecules encodes for a polypeptide as provided for herein.

[0018] In some embodiments, a host cell is provided. In some embodiments, the host cell comprises a nucleic acid molecule or molecules as provided for herein.

[0019] In some embodiments, a method of producing a polypeptide as provided for herein is provided, the method comprising culturing a host cell as provided for herein under conditions to express and produce the polypeptide.

[0020] In some embodiments, a method of treating cancer or an auto-immune disease in a subject is provided, the method comprising administering to the subject a pharmaceutical composition comprising a polypeptide as provided for herein.

[0021] In some embodiments, a method of reducing CD5 surface expression on an immune cell is provided, the method comprising contacting the immune cell expressing CD5 on its surface with a pharmaceutical composition comprising the polypeptide as provided for herein.

[0022] In some embodiments, a method of activating a T-cell is provided, the method comprising contacting a T cell expressing CD5 on its surface with a pharmaceutical composition comprising a polypeptide as provided for herein.

[0023] -2- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT

[0024] In some embodiments, a T-cell is provided, the T-cell comprising a chimeric antigen receptor and a polypeptide expressed on the surface of the T-cell, wherein the polypeptide expressed on the surface of the T-cell comprises an antigen binding domain that binds to a surface protein of the T-cell.

[0025] BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 illustrates graphical representations of non-limiting embodiments of polypeptides provided for herein.

[0026] FIG. 2 illustrates graphical representations of non-limiting embodiments of polypeptides provided for herein.

[0027] FIG. 3 illustrates the amino acid sequence and the domains of CD5, including the extracellular domains, the transmembrane domain, and the cytoplasmic domain and the boundaries of each.

[0028] FIG. 4A, 4B, and 4C illustrate the enhancement effect of tethered anti-CD5 antibodies on effector function of CART cells with surface CD5 expression as compared to soluble anti-CD5 antibodies. FIG. 4A illustrates the effect of soluble and tethered UCHT2 antibody on IFNy production. FIG. 4B illustrates the effect of soluble and tethered H65 antibody on IFNy production. FIG. 4C illustrates the effect of soluble and tethered isotype control on IFNy production.

[0029] FIG. 5 illustrates the enhancement effect of tethered anti-CD5 and / or anti-CD7 antibodies on effector function of CART cells with surface CD5 expression as compared to soluble anti-CD5 antibodies.

[0030] FIG. 6 illustrates the synergistic effect of tethered anti-CD5 and anti-CD7 antibodies on CAR-T cells activation.

[0031] FIG. 7 illustrates the synergistic effect of tethered anti-CD5 and anti-CD7 antibodies on CAR-T cells activation where the T cells have been stimulated with the antigen recognized by the CAR.

[0032] FIG. 8 illustrates that tethered anti-CD5 antibodies induce surface CD5 downregulation compared to control antibodies.

[0033] FIG. 9A and 9B illustrate that cross-linked anti-CD5 antibodies induce surface CD5 downregulation. FIG. 9A illustrates the control conditions. FIG. 9B illustrates the cross linked antibody conditions.

[0034] -3- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT

[0035] FIG. 10 illustrates that certain anti-CD5 scFv constructs expressed on the surface of T cells induces downregulation of surface CD5 in the same T cells.

[0036] FIG. 11 illustrates the transduction efficiency of anti-CD5 scFv and CAR19 constructs of FIG. 10.

[0037] FIG. 12 illustrates that the anti-CD5 scFv capable of down regulation of surface CD5 expression has no effect on expression of the CAR19 construct when co-expressed.

[0038] FIG. 13A and 13B illustrate the efficiencies of certain anti-CD5 antibodies in driving surface CD5 internalization in CART cells. FIG. 13A illustrates internalization as measured by BLla binding. FIG. 13B illustrates internalization as measured by UCHT2 binding.

[0039] FIG. 14A and 14B illustrate the enhancement effect of tethered anti-CD3 antibodies on PBMC cell stimulation as compared to soluble anti-CD3 antibodies. FIG. 14A illustrates the effect of soluble and tethered OKT3 antibody on IFNy production in donor D267. FIG. 14B illustrates the effect of soluble and tethered OKT3 antibody on IFNy production in donor D419.

[0040] FIG. 15 illustrates the synergistic effect of anti-CD3 antibody OKT3 in combination with various anti-CD5 antibodies on PMBC activation.

[0041] FIG. 16 A and 16B illustrate the efficiencies of additional anti-CD5 antibodies in driving surface CD5 internalization in CART cells. FIG. 16A illustrates internalization as measured by BLla binding. FIG. 16B illustrates internalization as measured by UCHT2 binding. FIG. 17A and 17B illustrate the synergistic effect of anti-CD3 antibody OKT3 in combination with various anti-CD5 antibodies on PMBC activation. FIG. 17A illustrates results with donor D780-P09. FIG. 17B illustrates results with donor D1103-P02.

[0042] DETAILED DESCRIPTION

[0043] As used herein and unless otherwise indicated, the term “about” means that the numerical value is approximate and small variations would not significantly affect the practice of the disclosed embodiment. Where a numerical limitation is used, unless indicated otherwise by the context, “about” means the numerical value can vary by ±10% and remain within the scope of the disclosed embodiments.

[0044] As used herein and in the appended claims, the singular forms “a”, “an” and “the” include plural reference unless the context clearly dictates otherwise.

[0045] -4- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT

[0046] As used herein, the term “animal” includes, but is not limited to, humans and nonhuman vertebrates such as wild, domestic, and farm animals. Accordingly, as used herein, the term “mammal” means a rodent (i.e., a mouse, a rat, or a guinea pig), a monkey, a cat, a dog, a cow, a horse, a pig, or a human. In some embodiments, the mammal is a human.

[0047] As used herein, the term “contacting” means bringing together of two elements in an in vitro system or an in vivo system. For example, “contacting” a therapeutic compound with an individual or patient or cell includes the administration of the compound or composition to an individual or patient, such as a human, as well as, for example, introducing a compound into a sample containing a cellular or purified preparation containing target.

[0048] As used herein, the terms “comprising” (and any form of comprising, such as “comprise”, “comprises”, and “comprised”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”), or “containing” (and any form of containing, such as “contains” and “contain”), are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. Any composition or method that recites the term “comprising” should also be understood to also describe such compositions as consisting, consisting of, or consisting essentially of the recited components or elements.

[0049] As used herein, the term “chimeric antigen receptor” or “CAR,” as used herein, refers to an artificial immune cell, such as but not limited to T-cell, receptor that is engineered to be expressed on an immune effector cell. The CAR can be incorporated into a cell, which can then be referred to as a “CAR-T cell” or other similar name. The CAR can have an antigen binding domain that can bind to a tumor antigen, such as CD 19, CD20, CD5, CD2, CD7, BCMA, and the like or as otherwise provided for herein. These are non-limiting examples. The T cells can be made in vivo or ex-vivo as provided for herein and known in the art. In some embodiments, the CAR may also comprise an intracellular activation domain, a transmembrane domain and an extracellular domain comprising a tumor associated antigen binding region. In some aspects, the CAR can comprise an extracellular domain comprising an anti-B cell binding domain fused to CD3-zeta transmembrane and intracellular domain.

[0050] As used herein, the term “fused” or “linked” when used in reference to a protein or molecule having different domains or heterologous sequences means that the protein domains are part of the same peptide chain that are connected to one another with either peptide bonds or other covalent bonding. The domains or section can be linked or fused directly to one -5- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT

[0051] another or another domain or peptide sequence can be between the two domains or sequences and such sequences would still be considered to be fused or linked to one another.

[0052] As used herein, the term “individual,” “subject,” or “patient,” which can be used interchangeably, means any animal, including mammals, such as mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, such as humans.

[0053] As used herein, the term “inhibit” refers to a result, symptom, or activity being reduced as compared to the activity or result in the absence of the compound that is inhibiting the result, symptom, or activity. In some embodiments, the result, symptom, or activity, is inhibited by about, or, at least, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%. An result, symptom, or activity can also be inhibited if it is completely elimination or extinguished.

[0054] As used herein, the phrase “in need thereof’ means that the subject has been identified as having a need for the particular method or treatment. In some embodiments, the identification can be by any means of diagnosis. In any of the methods and treatments described herein, the subject can be in need thereof. In some embodiments, the subject is in an environment or will be traveling to an environment in which a particular disease, disorder, or condition is prevalent.

[0055] As used herein, the phrase “integer from X to Y” means any integer that includes the endpoints. For example, the phrase “integer from 1 to 5” means 1, 2, 3, 4, or 5.

[0056] As used herein, the term “position,” is meant to refer to a location in the sequence of a polypeptide. Positions may be numbered sequentially, or according to an established format, such as, but not limited to, the EU Index or numbering system based on Kabat's amino acid positions for antibodies or Fc domains.

[0057] In some embodiments, the term “therapeutic molecule” can be used interchangeably with “therapeutic compound,” “molecule,” or “therapeutic,” and refers to any polypeptide, or protein provided for herein.

[0058] " Specific binding" or "specifically binds to" or is "specific for" a particular antigen, target, or an epitope means binding that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar

[0059] -6- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT

[0060] structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target.

[0061] Specific binding for a particular antigen, target, or an epitope can be exhibited, for example, by an antibody having a KD for an antigen or epitope of at least about 10‘4M, at least about 10-5M, at least about 10’6 M, at least about 10’7M, at least about 10’8M, at least about 10’9M, alternatively at least about 10-10M, at least about 10-11M, at least about 10-12M, or greater, where KD refers to a dissociation rate of a particular antibody -target interaction. Typically, an antibody that specifically binds an antigen or target will have a KD that is, or at least, 2-, 4-, 5-, 10-, 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000-, or more times greater for a control molecule relative to the antigen or epitope.

[0062] In some embodiments, specific binding for a particular antigen, target, or an epitope can be exhibited, for example, by an antibody having a KA or Kafor a target, antigen, or epitope of at least 2-, 4-, 5-, 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- or more times greater for the target, antigen, or epitope relative to a control, where KA or Ka refers to an association rate of a particular antibody-antigen interaction.

[0063] As provided herein, the compounds and compositions provided for herein can be used in methods of treatment as provided herein. As used herein, the terms “treat,” “treated,” or “treating” mean both therapeutic treatment and prophylactic measures wherein the object is to slow down (lessen) an undesired physiological condition, disorder or disease, or obtain beneficial or desired clinical results. For purposes of these embodiments, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of extent of condition, disorder or disease; stabilized (i.e., not worsening) state of condition, disorder or disease; delay in onset or slowing of condition, disorder or disease progression; amelioration of the condition, disorder or disease state or remission (whether partial or total), whether detectable or undetectable; an amelioration of at least one measurable physical parameter, not necessarily discernible by the patient; or enhancement or improvement of condition, disorder or disease. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival, as applicable for a specific disease, as compared to expected survival if not receiving treatment.

[0064] Provided herein are compounds, such as antibodies, polypeptides or fusion proteins, e.g., that can be used as therapeutics that include two or more effector domains that bind to at least two different epitopes or two different proteins. If an antibody, or antibody-like

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[0066] molecules, the epitopes can be on the same antigen or on different antigens. In some embodiments the effector domain is a ligand that binds to the target protein. In some embodiments, the effector domain is an antibody or antibody-like molecule that binds to the target protein. In some embodiments, the effector domain can be an antibody, antibody-like molecule, a ligand, a fibronectin domain (e.g. FN3), DARPIN, and the like.

[0067] In some embodiments, the polypeptide comprises a first effector domain and a second effector domain. The effector domain can also be referred to as a binding domain and should be considered equivalent terms and, as such, can be used interchangeably. In some embodiments, the polypeptide comprises a third effector domain, which can also be referred to as a binding domain.

[0068] The effector domains (binding domains) can be linked to one another through peptide or chemical linkers or through a Fc constant domain. The Fc domain can be wild-type or effectorless. Mutations that cause the Fc domain to be effectorless are known in art. For example, the mutation can be what is referred to as the “LALA”. The LALA mutations are L234A / L235A mutations, which are described in Lund J, Winter G, Jones PT, Pound JD, Tanaka T, Walker MR et al. Human Fc gamma RI and Fc gamma RII interact with distinct but overlapping sites on human IgG. J Immunol. 1991;147:2657-2662; and Wilkinson et al, Fc-engineered antibodies with immune effector functions completely abolished, PLoS One.

[0069] 2021; 16(12): e0260954, each of which are hereby incorporated by reference in its entirety. The numbering of residues in the Fc domain is according to the EU index, which is provided for herein and can be found in Kabat et al. (1991) Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, MD, and according to FIGs.3c-3f of U. S. Pat. App. Pub. No.2008 / 0248028. The “EU index” may also be referred to as “EU numbering”.

[0070] Another Fc mutation that can render the Fc domain effectorless or with reduced binding to the Fc receptors is L234A / L235A / P329G (LALAPG) or L234A / L235A / G237A. In some embodiments, the Fc receptor may have specific binding for FcyRIIa or FcyRIIb. Such mutations are known in the art and can also be incorporated into the polypeptides provided for herein.

[0071] The Fc can be of any isotype. As used herein, "isotype" refers to the immunoglobulin class (e.g., IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD, and IgE antibody) that is encoded by the heavy chain constant domain genes. The full-length amino acid sequence of each wild type human IgG constant region (including all domains, i.e., CHI domain, hinge, CH2

[0072] -8- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT

[0073] domain, and CH3 domain) is cataloged in the UniProt database available on-line, e.g., as

[0074] P01857 (IgGl), P01859 (IgG2), P01860 (IgG3), and P01861 (IgG4), or different allotypes thereof (SEQ ID NOs: 1, 2, 3, and 4, respectively). As used herein, a domain of a heavy chain constant region, e.g., the hinge, is of an " IgGl isotype," " IgG2 isotype," " IgG3 isotype," or

[0075] " IgG4 isotype," if the domain comprises the amino acid sequence of the corresponding

[0076] domain of the respective isotype, or a variant thereof (that has a higher homology to the corresponding domain of the respective isotype than it does to that of the other isotypes).

[0077] “Allotype” refers to naturally occurring variants within a specific isotype group,

[0078] which variants differ in a few amino acids (see, e.g., Jefferies et al. (2009) mAbs 1:1).

[0079] Molecules described herein may be of any allotype.

[0080] A “wild-type” protein or portion thereof is a version of the protein as it is found in nature. An amino acid sequence of a wild-type protein, e.g., a heavy chain constant region, is the amino acid sequence of the protein as it occurs in nature. Due to allotypic differences,

[0081] there can be more than one amino acid sequence for a wild-type protein. For example, there are several allotypes of naturally occurring human IGgl heavy chain constant regions (e.g., Jeffries et al. (2009) mAbs 1:1).

[0082] An immunoglobulin may be from any of the commonly known isotypes, including but not limited to IgA, secretory IgA, IgG and IgM. The IgG isotype is divided in subclasses in certain species: IgGl, IgG2, IgG3 and IgG4 in humans, and IgGl, IgG2a, IgG2b and IgG3 in mice. In certain embodiments, the antibodies described herein are of the human IgGl or IgG2 subtype. Immunoglobulins, e.g., human IgGl, exist in several allotypes, which differ from

[0083] each other in at most a few amino acids.

[0084] In some embodiments, the IgG proteins (hinge region underlined) are as provided in Table 1.

[0085] Table 1

[0086] Isotype Sequence

[0087] IgGl ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSS SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 1)

[0088] IgG2 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSS NFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHE DPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 2 )

[0089] lgG3 ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSS

[0090] SLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPK

[0091]

[0092] SCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVQFKWYVDGVEVHNAKTKPR -9- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT

[0093] EEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKS LSLSPGK (SEQ ID NO: 3)

[0094] IgG4 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSS SLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSQ EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQ PREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSR

[0095]

[0096] WQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 4)

[0097] An " Fc polypeptide" (fragment crystallizable region), " Fc domain", " Fc", or “constant domain” or an antibody refers to the C- terminal region of the heavy chain of an antibody that mediates the binding of the immunoglobulin to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or to the first component (Clq) of the classical complement system. Thus, an Fc polypeptide of an

[0098] antibody of isotype IgG comprises the heavy chain constant region of the antibody excluding the first constant region immunoglobulin domain (CHI). In IgG, IgA and IgD antibody

[0099] isotypes, the Fc polypeptide comprises CH2 and CH3 constant domains in each of the

[0100] antibody's two heavy chains; IgM and IgE Fc polypeptides comprise three heavy chain

[0101] constant domains (CH domains 2-4) in each polypeptide chain. For IgG, the Fc polypeptide comprises immunoglobulin domains consisting of the hinge, CH2 and CH3. For purposes herein, the Fc polypeptide is defined as starting at amino acid 216 and ending at amino acid

[0102] 447, wherein the numbering is according to the EU index as in Kabat. Kabat et al. (1991) Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda,

[0103] MD, and according to FIGs.3c-3f of U. S. Pat. App. Pub. No.2008 / 0248028. The “EU index” may also be referred to as “EU numbering”. In some embodiments, the Fc polypeptide

[0104] comprises the hinge region. The Fc may be a native (or naturally-occurring or wild-type) Fc, including any allotypic variant, or a variant Fc (e.g.. a non- naturally occurring Fc),

[0105] comprising, e.g., 1, 2, 3, 4, 5, 1-5, 1-10 or 5-10 or more amino acid mutations, e.g., substitutions, additions or deletions. For example, a variant Fc may comprise an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a wild-type Fc. Modified or mutated Fes may have enhanced or reduced effector function

[0106] and / or half-life. Fc may refer to this region in isolation or in the context of an Fc-comprising protein polypeptide such as a “binding protein comprising an Fc polypeptide,” also referred to as an “Fc fusion protein” (e.g., an antibody or immunoadhesion). In some embodiments, modified or variant Fc molecules have enhanced binding to FcyRIip.

[0107] A "hinge", "hinge domain" or "hinge region" or "antibody hinge region" refers to the domain of a heavy chain constant region that joins the CHI domain to the CH2 domain and

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[0109] includes the upper, middle, and lower portions of the hinge (Roux et al. J. Immunol.1998 161:4083). The hinge provides varying levels of flexibility between the binding and effector regions of an antibody and also provides sites for intermolecular disulfide bonding between the two heavy chain constant regions. The term “hinge” includes wild-type hinges (such as those set forth in Table 2), as well as variants thereof (e.g., non-naturally-occurring hinges or modified hinges). For example, the term “IgGl hinge” includes wild-type IgGl hinge, as shown below, and variants having 1, 2, 3, 4, 5, 1-3, 1-5, 3-5 and / or at most 5, 4, 3, 2, or 1 mutations, e.g., substitutions, deletions or additions. In some embodiments, the hinge regions are as provided in Table 2.

[0110] Table 2

[0111] Isotype Hinge Sequence

[0112] IgGl EPKSCDKTHTCPPCPAPELLGGP (SEQ ID NO: 10 )

[0113] IgG2 ELKTPLGDTTHTCPRCPAPELLGGP (SEQ ID NO: 11)

[0114] IgG3 ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPEL LGGP (SEQ ID NO: 12 )

[0115] IgG4 ESKYGPPCPSCPAPEFLGGP (SEQ ID NO: 13)

[0116]

[0117] The term “CHI domain” refers to the heavy chain constant region linking the variable domain to the hinge in a heavy chain constant domain. As used herein, a CHI domain includes wild type CHI domains, as well as variants thereof (e.g., non-naturally-occurring CHI domains or modified CHI domains). As used herein, CHI domain includes amino acid residues 1-98 of IgGl; 1-98 of IgG2; 1-98 of IgG3; and 1-98 of IgG4. For example, the term “CHI domain” includes wild-type CHI domains and variants thereof having 1, 2, 3, 4, 5, 1-3, 1-5, 3-5 and / or at most 5, 4, 3, 2, or 1 mutations, e.g., substitutions, deletions or additions.

[0118] The term “CH2 domain” refers to the heavy chain constant region linking the hinge to the CH3 domain in a heavy chain constant domain. As used herein, a CH2 domain includes wild-type CH2 domains, as well as variants thereof (e.g., non-naturally-occurring CH2 domains or modified CH2 domains). As used herein, CH2 domain includes amino acid residues 111-223 of IgGl; 111-219 of IgG2; 161-270 of IgG3; and 111-220 of IgG4. For example, the temi“CH2 domain” includes wild-type CH2 domains and variants thereof having 1, 2, 3, 4, 5, 1-3, 1-5, 3-5 and / or at most 5, 4, 3, 2, or 1 mutations, e.g., substitutions, deletions or additions.

[0119] The term “CH3 domain” refers to the heavy chain constant region that is C-terminal to the CH2 domain in a heavy chain constant domain. As used herein, a CH3 domain includes -11- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT

[0120] wild-type CH3 domains, as well as variants thereof (e.g., non-naturally- occurring CH3 domains or modified CH3 domains). As used herein, CH3 domain includes amino acid residues 224-330 of IgGl; 220-326 of IgG2; 271-376 of IgG3; and 226-322 of IgG4. For example, the term“CH3 domain” includes wild-type CH3 domains and variants thereof having 1, 2, 3, 4, 5, 1-3, 1-5, 3-5 and / or at most 5, 4, 3, 2, or 1 mutations, e.g., substitutions, deletions or additions.

[0121] The Fc domain or molecule can be a variant Fc molecules comprising variant Fc domains.

[0122] In some embodiments, a variant Fc molecule comprises a mutation that confers selective binding to FcγRIIβ over FcγRIIα. As used herein, in reference to FcyRIip, the term “selective binding” means that the Fc polypeptide binds preferentially to FcyRIip over FcyRIIa, that is with a higher affinity to FcyRIip over FcyRIIa. Examples of such mutations are provided in, for example, US 7662926, US 7655229, US 2009 / 0087428, US 10919952, US 2007 / 0253948, and US 2006 / 0073142, each of which is hereby incorporated by reference in its entirety, including the specific mutations that are descried that affect FcγRIIβ binding. In some embodiments, the mutation is as described in Shields et al., J. Biol. Chem. 2001, 276:6591-6604, which is hereby incorporated by reference in its entirety.

[0123] In some embodiments, the Fc polypeptide comprises a mutation that corresponds to S298A, E333A, or K334A, or any combination thereof. In some embodiments, the Fc polypeptide comprises the mutations of S298A, E333A, and K334A. In some embodiments, the mutations correspond to G236A, I332E, G236A, S239D, or I332E, or any combination thereof. In some embodiments, the Fc polypeptide comprises the mutations of G236A, I332E, G236A, S239D, and I332E. The mutations can also be as provided for in, Richards et al., Mol Cancer Ther 2008;7(8). August 2008, which is hereby incorporated by reference in its entirety, including the specific mutations that are descried that affect the FcγRIIb or FcγRIIa binding. In some embodiments, the Fc polypeptide comprises a N235S or L328F mutation. In some embodiments, the Fc polypeptide comprises a N235S and L328F mutation. The mutations can also be as provided for in Shang et al., The Journal of Biological Chemistry VOL. 289, NO. 22, pp. 15309-15318, May 30, 2014, which is hereby incorporated by reference in its entirety, including the specific mutations that are descried that affect the FcγRIIb or FcγRIIa binding.

[0124] In some embodiments, the Fc mutation is as described in U. S. Patent No. 10,618,965; EP Serial No. 2679681; EP Serial No. 3604330, US 2014 / 0093496, US 2015 / 0203577, U. S.

[0125] -12- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT

[0126] Patent No. 9,540,451, EP Serial No. 2331578; EP Serial No. 3190128; U. S. Patent No.

[0127] 9,902,773; EP. Serial No. 3342782, U. S. Publication No. 2020 / 0332024; EP Serial No.

[0128] 2796469; EP Serial No. 2331578; EP Serial No. 3190128; U. S. Patent No. 9,902,773, EP Serial No. 2331578; EP Serial No. 3190128, U. S. Patent No. 9,493,578, U. S. Patent No. 9,394,366, U. S. Patent No. 9,914,778, EP Serial No. 2940043, US 9,890,218, EP Serial No.

[0129] 2940135; U. S. Patent No. 10,766,960, U. S. Patent No. 10,919,953, EP 3721900, EP2889377, US 2016 / 0039912, EP 2982689, or EP 3783017, each of which is hereby incorporated by reference in its entirety, including the specific mutations that are descried that affect the FcγRIIb or FcγRIIa binding.

[0130] In some embodiments, the Fc polypeptide comprises a mutation of P238D, which is described in Mimoto et al., Protein Eng Des Sel. 2013 Oct; 26(10): 589-598, which is incorporated by reference in its entirety.

[0131] In some embodiments, the Fc polypeptide comprises what is referred to as the “AAA” mutations, which are Leu234Ala, Leu235Ala, and Gly237Ala (EU numbering).

[0132] In some embodiments, the Fc polypeptide comprises what is referred to as the “LALA” mutations, which are Leu234Ala and Leu235Ala (EU numbering).

[0133] In some embodiments, the Fc polypeptide comprises at least one mutation that extends the half-life of the Fc polypeptide. In some embodiments, the at least one mutation that extends the half-life of the Fc polypeptide, is such as those known in the art, such as, without limitation, a set of mutations of M428L and N434S (“LS” mutations), or M252Y, S254T, and T256E (“YTE” mutations) mutations. The extension mutations can be combined with or used independently of the other Fc mutations provided for herein, such as those that provide selective binding to FcγRIIB or those that impair the function of the Fc polypeptide or that make the Fc polypeptide effectorless, such as “AAA” or “LALA”.

[0134] In some embodiments, the mutations in the Fc polypeptide, which is according to the known numbering system (EU numbering), are selected from the group consisting of: L234A, L235A, L234F, L235E, P329G, P331S, N297A, N297G, N297Q, G236A, A330S, S239D, I332E, S267E, H268F, S324T, Y296W, T299A, V308P, H310A, R409K, Y435H, T307A, T309A, T309K, K322A, K326W, K334W, K326A, K334A, G237A, P238S, H268A, or any combination thereof.

[0135] The mutations and positions of the Fc polypeptide, which can also be referred to as the Fc polypeptide, are according to EU numbering.

[0136] -13- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT

[0137] As used herein, a Fc polypeptide / domain comprising a mutation at a specific position is as compared to the wild-type Fc according the numbering system (EU numbering) as referenced herein.

[0138] Examples of peptide linkers that can be used are known in the art and non-limiting examples are provide for herein.

[0139] Antibody molecule, as used herein, refers to a polypeptide, e.g., an immunoglobulin chain or fragment thereof, comprising at least one functional immunoglobulin variable domain sequence. An antibody molecule encompasses antibodies (e.g., full-length antibodies) and antibody fragments. In some embodiments, an antibody molecule comprises an antigen binding or functional fragment of a full length antibody, or a full length immunoglobulin chain. For example, a full-length antibody is an immunoglobulin (Ig) molecule (e.g., an IgG antibody) that is naturally occurring or formed by normal immunoglobulin gene fragment recombinatorial processes). In embodiments, an antibody molecule refers to an immunologically active, antigen-binding portion of an immunoglobulin molecule, such as an antibody fragment. An antibody fragment, e.g., functional fragment, comprises a portion of an antibody, e.g.. Fab, Fab', F(ab')2, F(ab)2, variable fragment (Fv), domain antibody (dAb), or single chain variable fragment (scFv). A functional antibody fragment binds to the same antigen as that recognized by the intact (e.g., full-length) antibody. The terms “antibody fragment” or “functional fragment” also include isolated fragments consisting of the variable regions, such as the “Fv” fragments consisting of the variable regions of the heavy and light chains or recombinant single chain polypeptide molecules in which light and heavy variable regions are connected by a peptide linker (“scFv proteins”). In some embodiments, an antibody fragment does not include portions of antibodies without antigen binding activity, such as Fc fragments or single amino acid residues. Exemplary antibody molecules include full length antibodies and antibody fragments, e.g., dAb (domain antibody), single chain, Fab, Fab’, and F(ab’)2 fragments, and single chain variable fragments (scFvs).

[0140] The term “antibody molecule” also encompasses whole or antigen binding fragments of domain, or single domain, antibodies, which can also be referred to as “sdAb” or “VHH.” Domain antibodies comprise either VH or VL that can act as stand-alone, antibody fragments. Additionally, domain antibodies include heavy-chain-only antibodies (HCAbs). Domain antibodies also include a CH2 domain of an IgG as the base scaffold into which CDR loops are grafted. It can also be generally defined as a polypeptide or protein comprising an amino acid sequence that is comprised of four framework regions interrupted by three complementarity determining regions. This is represented as FR1- CDR1 -FR2-CDR2-FR3- -14- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT

[0141] CDR3-FR4. sdAbs can be produced in camelids such as llamas, but can also be synthetically generated using techniques that are well known in the art. The numbering of the amino acid residues of a sdAb or polypeptide is according to the general numbering for VH domains given by Kabat et al. (" Sequence of proteins of immunological interest," US Public Health Services, NIH Bethesda, MD, Publication No. 91, which is hereby incorporated by reference). According to this numbering, FR1 of a sdAb comprises the amino acid residues at positions 1-30, CDR1 of a sdAb comprises the amino acid residues at positions 31-36, FR2 of a sdAb comprises the amino acids at positions 36-49, CDR2 of a sdAb comprises the amino acid residues at positions 50-65, FR3 of a sdAb comprises the amino acid residues at positions 66- 94, CDR3 of a sdAb comprises the amino acid residues at positions 95-102, and FR4 of a sdAb comprises the amino acid residues at positions 103-113. Domain antibodies are also described in W02004041862 and WO2016065323, each of which is hereby incorporated by reference. The domain antibodies can be a targeting moiety as described herein.

[0142] The polypeptides provided for herein, which can be a combination of antibody molecules or antibody molecules and ligands, can be monospecific (e.g., monovalent or bivalent), bispecific (e.g., bivalent, trivalent, tetravalent, pentavalent, or hexavalent), trispecific (e.g., trivalent, tetravalent, pentavalent, hexavalent), or with higher orders of specificity (e.g, tetraspecific) and / or higher orders of valency beyond hexavalency. Even if the polypeptide is monospecific for a target protein it may have different binding domains that bind to different epitopes of the same antigen. An antibody molecule can comprise a functional fragment of a light chain variable region and a functional fragment of a heavy chain variable region, or heavy and light chains may be fused together into a single polypeptide.

[0143] Effector, as that term is used herein, refers to a molecule, e.g., a soluble or cell surface molecule, which mediates an immune response, such as an activation signal or an inhibition signal of an immune cells. In some embodiments, the effector is an antibody, which can be referred to as an effector domain or binding domain. In some embodiments, the effectors binding domains as provided for herein, refers to a polypeptide (e.g.) that has sufficient binding specificity that it can bind the effector with sufficient specificity that it can serve as an effector binding / modulating molecule. In some embodiments, it binds to effector with at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95% of the affinity of the naturally occurring counter-ligand. In some embodiments, it has at least 60, 70, 80, 90, 95, 99, or 100% sequence identity,or substantial sequence identity, with a naturally occurring counter-ligand for the -15- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT

[0144] effector. It can mediate the signal by either enhancing or inhibiting signal by agonizing or antagonizing the binding partner’s function, but can also mediate the signal by leading to internalization or turnover of the target molecule. For example, polypeptides provided for herein can bind to CD5, such as the extracellular domain of CD5, which has a plurality of domains. Without being bound to any particular theory, when an antibody binding domain binds to an extracellular domain of CD5, it can lead to the internalization of CD5, which then does not allow CD5 to bind to its cognate ligand, which can be used to antagonize the function of CD5.

[0145] The domains can have similarity to those sequences or molecules provided for herein or those that are incorporated by reference. Sequence identity, percentage identity, and related terms, as those terms are used herein, refer to the relatedness of two sequences, e.g., two nucleic acid sequences or two amino acid or polypeptide sequences. In the context of an amino acid sequence, the term "substantially identical" is used herein to refer to a first amino acid that contains a sufficient or minimum number of amino acid residues that are i) identical to, or ii) conservative substitutions of aligned amino acid residues in a second amino acid sequence such that the first and second amino acid sequences can have a common structural domain and / or common functional activity. For example, amino acid sequences that contain a common structural domain having at least about 85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a reference sequence, e.g., a sequence provided herein.

[0146] In the context of nucleotide sequence, such as those encoding for the domains, the term "substantially identical" is used herein to refer to a first nucleic acid sequence that contains a sufficient or minimum number of nucleotides that are identical to aligned nucleotides in a second nucleic acid sequence such that the first and second nucleotide sequences encode a polypeptide having common functional activity, or encode a common structural polypeptide domain or a common functional polypeptide activity. For example, nucleotide sequences having at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a reference sequence, e.g., a sequence provided herein.

[0147] The term “functional variant” refers to polypeptides that have a substantially identical amino acid sequence to the naturally-occurring sequence, or are encoded by a substantially identical nucleotide sequence, and are capable of having one or more activities of the naturally-occurring sequence.

[0148] Calculations of homology or sequence identity between sequences (the terms are used interchangeably herein) can be performed as follows.

[0149] -16- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT

[0150] To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology").

[0151] 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, which need to be introduced for optimal alignment of the two sequences.

[0152] The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453 ) algorithm which has been incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http: / / www.gcg.com), using a NWSgapdna. CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that should be used unless otherwise specified) are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

[0153] The percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4: 11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

[0154] The nucleic acid and protein sequences described herein can be used as a "query sequence" to perform a search against public databases to, for example, identify other family -17- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT

[0155] members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to for example any a nucleic acid sequence provided herein. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to protein molecules provided herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http: / / www.ncbi.nlm.nih.gov.

[0156] It is understood that the molecules and compounds of the present embodiments may have additional conservative amino acid substitutions.

[0157] 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. These families include amino acids with 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).

[0158] The present disclosure provides, for example, effector domains that can act as PD-1 agonists. Without being bound to any particular theory, agonism of PD-1 inhibits T cell activation / signaling and can be accomplished by different mechanisms. For example crosslinking can lead to agonism, bead-bound, functional PD-1 agonists have been described (Akkaya. Ph. D. Thesis: Modulation of the PD-1 pathway by inhibitory antibody superagonists. Christ Church College, Oxford, UK, 2012), which is hereby incorporated by reference. Crosslinking of PD-1 with two mAbs that bind non-overlapping epitopes induces PD-1 signaling (Davis, US 2011 / 0171220), which is hereby incorporated by reference.

[0159] Another example is illustrated through the use of a goat anti-PD-1 antiserum (e.g. AF1086, R& D Systems) which is hereby incorporated by reference, which acts as an agonist when soluble (Said et al., 2010, Nat Med) which is hereby incorporated by reference. Non-limiting examples of PD-1 agonists that can be used in the present embodiments include, but are not limited to, UCB clone 19 or clone 10, PD1 AB-1, PD1 AB-2, PD1 AB-3, PD1AB-4 and -18- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT

[0160] PD1AB-5, PD1AB-6 (Anaptys / Celgene), PD1-17, PD1-28, PD1-33 and PD1-35 (Collins et al, US 2008 / 0311117 Al).

[0161] Antibodies against PD-1 and uses therefor, which is incorporated by reference), or can be a bi-specific, monovalent anti-PD-1, and the like. In some embodiments, the PD-1 agonist antibodies can be antibodies that block binding of PD-L1 to PD-1. In some embodiments, the PD-1 agonist antibodies can be antibodies that do not block binding of PD-L1 to PD-1.

[0162] PD-1 is an Ig superfamily member expressed on activated T cells and other immune cells. The natural ligands for PD-1 appear to be PD-L1 and PD-L2. Without being bound to any particular theory, when PD-L1 or PD-L2 bind to PD-1 on an activated T cell, an inhibitory signaling cascade is initiated, resulting in attenuation of the activated T effector cell function. Thus, blocking the interaction between PD-1 on a T cell, and PD-L1 / 2 on another cell (eg tumor cell) with a PD-1 antagonist is known as checkpoint inhibition, and releases the T cells from inhibition. In contrast, PD-1 agonist antibodies can bind to PD-1 and send an inhibitory signal and attenuate the function of a T cell. Thus, PD-1 agonist antibodies can be incorporated into various embodiments described herein as an effector molecule binding / modulating moiety, which can accomplish localized tissue-specific immunomodulation when paired with a targeting moiety.

[0163] In some embodiments, the antibody is an anti-PD-1 antibody which binds to PD-1. In some embodiments, the antibody binds to amino acids of an epitope of PD-1.

[0164] In some embodiments, anti-PD-1 antibodies, such as those provided herein, bind to an epitope on PD-1. PD-1 is a type I membrane protein, which has the amino acid sequence as set forth in:

[0165] MQIPQAPWPWWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATF TCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGR DFHMSWRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVP TAHP SPSPRPAGQFQTLWGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQP LKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTS SPARRGSADGPRSAQPLRPEDGHCSWPL (SEQ ID NO: 14 )

[0166] In some embodiments, when the polypeptides provided for herein comprise an effector domain that binds to PD-1, it is an the antibody that binds to PD-1 that is an agonist. In some embodiments, the antibody that binds to PD-1 is an antagonist. Examples of anti-PDl antibodies are known and any PD-1 antibody can be linked to the binding domain that binds to CD5, such as an extracellular domain of CD5.

[0167] As provided for herein the different domains, molecules, or polypeptides can be linked together with a linker domain or region. Any linker region described herein can be -19- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT

[0168] used as a linker. Linkers can be for example, glycine / serine linkers. In some embodiments, the linker can comprise one or more repeats of GGGGS (SEQ ID NO: 15). In some embodiments, the linker comprises 1, 2, 3, 4, or 5 repeats. In some embodiments, the linker comprises GGGGSGGGGS (SEQ ID NO: 16). In some embodiments, the linker comprises GGGGSGGGGSGGGGS (SEQ ID NO: 17). In some embodiments, the linker comprises: GGGGS (SEQ ID NO: 15), (GGGGS)3 (SEQ ID NO: 17), (GGGGS)n (n=l, 2, 3. 4) (SEQ ID NO: 18). (Gly)8(SEQ ID NO: 19), (Gly)6(SEQ ID NO: 20), (EAAAK)3(SEQ ID NO: 21), (EAAK)n(n=1-3) (SEQ ID NO: 22), A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO: 23), or AEAAAKEAAAKA (SEQ ID NO: 24). These linkers can be used in any of the compounds, polypeptides or compositions provided herein. These peptide linkers are non-limiting examples and other peptide linkers can also be used.

[0169] In some embodiments, the polypeptide comprise the following (e.g., in N-terminus to C -terminus order or vice versa):

[0170] R1 — Linker Region A — R2

[0171] wherein, R1 and R2, each independently comprises an effector binding domain, e.g., anti-CD5 antibody, anti-immune cell surface protein antibody, or a ligand that binds to a immune cell surface protein. The Linker Region A can comprise moieties that can associate with one another, such as an Fc polypeptide. Thus, the polypeptide can form a dimer of such polypeptides. In some embodiments, the polypeptide can form a heterodimer with a polypeptide having formula of R3 — Linker Region B — R4, wherein R3 and R4 can each independently comprises an effector binding domain, e.g., anti-CD5 antibody, anti-immune cell surface protein antibody, or a ligand that binds to a immune cell surface protein and Linker Region B can comprise moieties that can associate with one another, such as an Fc polypeptide, provided that the two polypeptide chains are not identical, and therefore, form a heterodimer. They can be different in the linker region, such that the Fc polypeptides form a heterodimer or at least one of the effector domains of the polypeptide chains are different.

[0172] In some embodiments, the bispecific antibodies (that bind to either two different proteins or to different epitopes on the same protein) are comprised of four polypeptide chains comprising the following:

[0173] Chain 1: nt-VH1-CH1-CH2-CH3-Linker A-scFv[VL2-Linker Region B-VH2]-ctChain 2: nt-VH1-CH1-CH2-CH3-Linker A-scFv[VL2-Linker Region B-VH2]-ct Chain 3: nt-VLl-CL-ct

[0174] Chain 4: nt-VLl-CL-ct.

[0175] -20- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT

[0176] In some embodiments, the bispecific antibodies are comprised of four polypeptide chains comprising the following:

[0177] Chain 1: nt-VHl -CH 1-CH2-CH3 -Linker A-scFv[VH2-Linker Region B-VL2]-ct Chain 2: nt-VHl -CH 1-CH2-CH3 -Linker A-scFv[VH2-Linker Region B-VL2]-ct Chain 3: nt-VLl-CL-ct

[0178] Chain 4: nt-VLl-CL-ct.

[0179] In some embodiments, the bispecific antibodies are comprised of four polypeptide chains that do not contain the Fc polypeptide, which can be illustrated as having the following formula:

[0180] Chain 1: nt-VHl -CHI -Linker A-scFv[VH2-Linker Region B-VL2]-ct

[0181] Chain 2: nt-VHl -CHI -Linker A-scFv[VH2-Linker Region B-VL2]-ct

[0182] Chain 3: nt-VLl-CL-ct

[0183] Chain 4: nt-VLl-CL-ct.

[0184] In some embodiments, the bispecific antibodies are comprised of four polypeptide chains that do not contain the Fc polypeptide, which can be illustrated as having the following formula:

[0185] Chain 1: nt-VHl— Linker A-scFv[VH2-Linker Region B-VL2]-ct

[0186] Chain 2: nt-VHl -Linker A-scFv[VH2-Linker Region B-VL2]-ct

[0187] Chain 3: nt-VLl-CL-ct

[0188] Chain 4: nt-VLl-CL-ct.

[0189] The Fc domain can be effectorless or can be an Fc domain that selectively binds to FcyRIIB or FcyRIIA, such as those provided for herein.

[0190] In some embodiments, the bispecific antibodies are comprised of four polypeptide chains that do not contain the Fc polypeptide, which can be illustrated as having the following formula:

[0191] Chain 1: nt-VHl -CHI -Linker A-scFv[VL2-Linker Region B-VH2]-ct

[0192] Chain 2: nt-VHl -CHI -Linker A-scFv[VL2-Linker Region B-VH2]-ct

[0193] Chain 3: nt-VLl-CL-ct

[0194] Chain 4: nt-VLl-CL-ct.

[0195] In some embodiments, the bispecific antibodies are comprised of four polypeptide chains that do not contain the Fc polypeptide, which can be illustrated as having the following formula:

[0196] Chain 1: nt- VH 1 -Linker A-scFv[VL2-Linker Region B-VH2]-ct

[0197] -21- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT

[0198] Chain 2: nt-VHl-Linker A-scFv[VL2-Linker Region B-VH2]-ct

[0199] Chain 3: nt-VLl-CL-ct

[0200] Chain 4: nt-VLl-CL-ct.

[0201] In some embodiments, the bispecific antibodies are comprised of four polypeptide chains comprising the following:

[0202] Chain 1: nt-VHl-CHl-CH2-CH3-ct

[0203] Chain 2: nt-VHl-CHl-CH2-CH3-ct

[0204] Chain 3: nt-VLl-CL-ct

[0205] Chain 4: nt-VLl-CL-ct.

[0206] In some embodiments, the bispecific antibodies are comprised of four polypeptide chains comprising the following:

[0207] Chain 1: nt-VHl-CHl-CH2-CH3-Linker A-scFv[VL2-Linker Region B-VH2]-ct Chain 2: nt-VHl-CHl-CH2-CH3-Linker A-scFv[VL2-Linker Region B-VH2]-ct Chain 3: nt-VLl-Ck-ct

[0208] Chain 4: nt-VLl-Ck-ct.

[0209] In some embodiments, the bispecific antibodies are comprised of four polypeptide chains comprising the following:

[0210] Chain 1: nt-VHl-CHl-CH2-CH3-Linker A-scFv[VH2-Linker Region B-VL2]-ct Chain 2: nt-VHl-CHl-CH2-CH3-Linker A-scFv[VH2-Linker Region B-VL2]-ct Chain 3: nt-VLl-Ck-ct

[0211] Chain 4: nt-VLl-Ck-ct.

[0212] In some embodiments, the bispecific antibodies are comprised of four polypeptide chains that do not contain the Fc polypeptide, which can be illustrated as having the following formula:

[0213] Chain 1: nt-VHl -CHI -Linker A-scFv[VH2-Linker Region B-VL2]-ct

[0214] Chain 2: nt-VHl -CHI -Linker A-scFv[VH2-Linker Region B-VL2]-ct

[0215] Chain 3: nt-VLl-Ck-ct

[0216] Chain 4: nt-VLl-Ck-ct.

[0217] In some embodiments, the bispecific antibodies are comprised of four polypeptide chains that do not contain the Fc polypeptide, which can be illustrated as having the following formula:

[0218] Chain 1: nt-VHl— Linker A-scFv[VH2-Linker Region B-VL2]-ct

[0219] Chain 2: nt-VHl-Linker A-scFv[VH2-Linker Region B-VL2]-ct

[0220] Chain 3: nt-VLl-Ck-ct

[0221] -22- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT

[0222] Chain 4: nt-VLl-Ck-ct.

[0223] The Fc domain can be effectorless or can be an Fc domain that selectively binds to FcyRIIB or FcyRIIA, such as those provided for herein.

[0224] In some embodiments, the bispecific antibodies are comprised of four polypeptide chains that do not contain the Fc polypeptide, which can be illustrated as having the following formula:

[0225] Chain 1: nt-VHl -CHI -Linker A-scFv[VL2-Linker Region B-VH2]-ct

[0226] Chain 2: nt-VHl -CHI -Linker A-scFv[VL2-Linker Region B-VH2]-ct

[0227] Chain 3: nt-VLl-Ck-ct

[0228] Chain 4: nt-VLl-Ck-ct.

[0229] In some embodiments, the bispecific antibodies are comprised of four polypeptide chains that do not contain the Fc polypeptide, which can be illustrated as having the following formula:

[0230] Chain 1: nt-VHl— Linker A-scFv[VL2-Linker Region B-VH2]-ct

[0231] Chain 2: nt-VHl-Linker A-scFv[VL2-Linker Region B-VH2]-ct

[0232] Chain 3: nt-VLl-Ck-ct

[0233] Chain 4: nt-VLl-Ck-ct.

[0234] In some embodiments, the bispecific antibodies are comprised of four polypeptide chains comprising the following:

[0235] Chain 1: nt-VHl -CH l-CH2-CH3-ct

[0236] Chain 2: nt-VHl-CHl-CH2-CH3-ct

[0237] Chain 3: nt-VLl-Ck-ct

[0238] Chain 4: nt-VLl-Ck-ct.

[0239] In some embodiments, the VH1 and VL1 form an antigen binding domain that binds to an extracellular domain of CD5. In some embodiments, the scFv binds to an extracellular domain of CD5. In some embodiments, the VH1 and VL1 bind to the protein present on the surface of an immune cell, such as, but not limited to, a T cell, B Cell, dendritic cell, or NK cell. In some embodiments, the scFv binds to the protein present on the surface of an immune cell. In some embodiments, the VH1 and VL1 form an antigen binding domain binds to a recombinant engineered protein or non-native sequence present on the surface of an immune cell. In some embodiments, the scFv form an antigen binding domain binds to a recombinant engineered protein or non-native sequence present on the surface of an immune

[0240] -23- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT

[0241] cell. Examples of such recombinant proteins are provided for herein, but should not be considered limiting.

[0242] In some embodiments, the CH1-CH2-CH3 comprises an amino acid sequence of any Fc provided herein.

[0243] In some embodiments, chains 1 and 2 are identical to each other, and chains 3 and 4 are identical to each other. In some embodiments, chains 3 and 4 are identical and chains 1 and 2 are different from one another or are different from one another at the N or C terminus or both. In some embodiments, each of the chains have different sequences. In some embodiments, wherein chain 1 forms a homodimer with chain 2; and chain 3 and 4 associate with chain 1 and chain 2. That is, when each light chain associates with each heavy chain, VL1 associates with VH1 and CL associates with CHI to form two functional Fab units. Without being bound to any particular theory, each scFv unit is intrinsically functional since VL2 and VH2 can be covalently linked in tandem with a linker as provided herein.

[0244] The sequences of Linker A and Linker Region B, which are independent of one another can be the same or different as provided for herein. In addition to having a Fc polypeptide or not, the linkers can independently comprise the glycine or short peptide linkers as provided for herein.

[0245] The scFv may also be arranged in the NT-VH2-VL2-CT or NT-VL2-VH2-CT orientation. NT or nt stands for N-terminus and CT or ct stands for C-terminus of the protein. In some embodiments, the CHI, CH2, and CH3 are the domains from the IgG Fc polypeptide, and CL stands for Constant Light chain, which can be either kappa or lambda family light chains. The other definitions stand for the way they are normally used in the art.

[0246] Another non-limiting example of a compound as provided for herein is illustrated in FIG. 1 and FIG.2. These illustrations are overall configurations and are not intended to represent the entire sequence, such as linkers. Other formats for multi-specific antibodies can be found in U. S. Patent No. 11,739,144 and U. S. Application Publication No. 20240052034, each of which is hereby incorporated by reference in its entirety. Although, these references describe various specific bispecific antibodies or antibody formats that do not include anti-CD5 antibodies or binding to other target proteins as provided for herein, the formats can be adapted for the polypeptides provided for herein.

[0247] Thus, the present application also provided for the following non-limiting embodiments.

[0248] In some embodiments, a polypeptide is provided comprising a first binding domain (effector moiety) and a second binding domain (effector moiety). In some embodiments, the -24- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT

[0249] first binding domain binds to an extracellular domain of CD5 and the second binding domain binds to an immune cell surface protein.

[0250] The full length sequence of CD5, before it is processed is as follows:

[0251] MPMGSLQPLATLYLLGMLVASCLGRLSWYDPDFQARLTRSNSKCQGQLEVYLKDGWHMVCSQ SWGRSSKQWEDPSQASKVCQRLNCGVPLSLGPFLVTYTPQSSIICYGQLGSFSNCSHSRNDM CHSLGLTCLEPQKTTPPTTRPPPTTTPEPTAPPRLQLVAQSGGQHCAGVVEFYSGSLGGTIS YEAQDKTQDLENFLCNNLQCGSFLKHLPETEAGRAQDPGEPREHQPLP I WKI NSSCTSLE HCFRKIKPQKSGRVLALLCSGFQPKVQSRLVGGSSICEGTVEVRQGAQWAALCDSSSARSSL RWEEVCREQQCGSVNSYRVLDAGDPTSRGLFCPHQKLSQCHELWERNSYCKKVFVTCQDPNP AGLAAGTVASIILALVLLWLLWCGPLAYKKLVKKFRQKKQRQWIGPTGMNQNMSFHRNHT ATVRSHAENPTASHVDNEYSQPPRNSHLSAYPALEGALHRSSMQPDNSSDSDYDLHGAQRL

[0252] (SEQ ID NO: 5 )

[0253] The CD5 sequence when processed cleaves the leader sequence (first 24 amino acid residues) and leaves a mature sequence of:

[0254] RLSWYDPDFQARLTRSNSKCQGQLEVYLKDGWHMVCSQSWGRSSKQWEDPSQASKVCQRLNC GVPLSLGPFLVTYTPQSS I ICYGQLGSFSNCSHSRNDMCHSLGLTCLEPQKTTPPTTRPPPT TTPEPTAPPRLQLVAQSGGQHCAGWEFYSGSLGGTISYEAQDKTQDLENFLCNNLQCGSFL KHLPETEAGRAQDPGEPREHQPLPIQWKIQNSSCTSLEHCFRKIKPQKSGRVLALLCSGFQP KVQSRLVGGSSICEGTVEVRQGAQWAALCDSSSARSSLRWEEVCREQQCGSVNSYRVLDAGD PTSRGLFCPHQKLSQCHELWERNSYCKKVFVTCQDPNPAGLAAGTVAS I ILALVLLWLLW CGPLAYKKLVKKFRQKKQRQWIGPTGMNQNMSFHRNHTATVRSHAENPTASHVDNEYSQPPR NSHLSAYPALEGALHRSSMQPDNSSDSDYDLHGAQRL (SEQ ID NO: 6)

[0255] Residues 25-372 (shown with underline) of the full length sequence or 1-348 (shown with underline) of the mature sequence comprise the extracellular domain, or the domain that is exposed on the surface of the cells expressing CD5. In some embodiments, the first binding domain binds to an extracellular region of CD5, such as those illustrated in FIG. 3. FIG. 3 illustrates the domains of DI, DII, and Dill of CD5 and the amino acid residue boundaries of each. The amino acid boundaries illustrated in FIG. 3 are in reference to SEQ ID NO: 5. FIG. 3 is reproduced from Rodamilans, et al., The Journal Of Biological Chemistry VOL. 282, NO. 17, pp. 12669 -12677, April 27, 2007, which is hereby incorporated by reference in its entirety. The boundaries in reference to SEQ ID NO: 6 would be 25 less than what is illustrated in FIG. 3 due to the deletion of the leader sequence. Thus, in some embodiments, the first binding domain binds to such extracellular domain of CD5. In some embodiments, the first binding domain binds to domain DI, DII, or Dill of the extracellular domain of CD5. In some embodiments, the first binding domain binds to an epitope comprising residues from the region of residues 35-133 of SEQ ID NO: 5 (Domain DI) or the corresponding sequence of SEQ ID NO: 6. In some embodiments, the first binding domain binds to an epitope -25- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT

[0256] comprising residues from the region of residues 165-268 of SEQ ID NO: 5 (Domain DII) or the corresponding sequence of SEQ ID NO: 6. In some embodiments, the first binding domain binds to an epitope comprising residues from the region of residues 276-368 of SEQ ID NO: 5 (Domain Dill) or the corresponding sequence of SEQ ID NO: 6. In some embodiments, first binding domain binds to an epitope comprising a residue from the region of residues 25-35 of SEQ ID NO: 5, or the corresponding sequence of SEQ ID NO: 6. In some embodiments, first binding domain binds to an epitope comprising a residue from the region of residues 133-165 of SEQ ID NO: 5, or the corresponding sequence of SEQ ID NO: 6. In some embodiments, first binding domain binds to an epitope comprising a residue from the region of residues 268-276 of SEQ ID NO: 5, or the corresponding sequence of SEQ ID NO: 6. In some embodiments, first binding domain binds to an epitope comprising a residue from the region of residues 368-380 of SEQ ID NO: 5, or the corresponding sequence of SEQ ID NO: 6.

[0257] In some embodiments, the second binding domain binds to an extracellular domain of CD5. In some embodiments, the second binding domain binds to domain DI, DII, or Dill of the extracellular domain of CD5. In some embodiments, the second binding domain binds to an epitope comprising residues from the region of residues 35-133 of SEQ ID NO: 5 (Domain DI) or the corresponding sequence of SEQ ID NO: 6. In some embodiments, the second binding domain binds to an epitope comprising residues from the region of residues 165-268 of SEQ ID NO: 5 (Domain DII) or the corresponding sequence of SEQ ID NO: 6. In some embodiments, the second binding domain binds to an epitope comprising residues from the region of residues 276-368 of SEQ ID NO: 5 (Domain Dill) or the corresponding sequence of SEQ ID NO: 6. In some embodiments, second binding domain binds to an epitope comprising a residue from the region of residues 25-35 of SEQ ID NO: 5, or the corresponding sequence of SEQ ID NO: 6. In some embodiments, second binding domain binds to an epitope comprising a residue from the region of residues 133-165 of SEQ ID NO: 5, or the corresponding sequence of SEQ ID NO: 6. In some embodiments, second binding domain binds to an epitope comprising a residue from the region of residues 268-276 of SEQ ID NO: 5, or the corresponding sequence of SEQ ID NO: 6. In some embodiments, second binding domain binds to an epitope comprising a residue from the region of residues 368-380 of SEQ ID NO: 5, or the corresponding sequence of SEQ ID NO: 6.

[0258] In some embodiments, the first binding domain and the second binding domain binds to different CD5 epitopes. In some embodiments, the first binding domain and the second binding domain binds to the same CD5 epitope.

[0259] -26- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT

[0260] In some embodiments, i) the first binding domain binds: to domain DI, DII, or Dill of the extracellular domain of CD5; to an epitope comprising residues from the region of residues 35-133 of SEQ ID NO: 5 (Domain DI), or the corresponding sequence of SEQ ID NO: 6; to an epitope comprising residues from the region of residues 165-268 of SEQ ID NO: 5 (Domain DII), or the corresponding sequence of SEQ ID NO: 6; to an epitope comprising residues from the region of residues 276-368 of SEQ ID NO: 5 (Domain Dill), or the corresponding sequence of SEQ ID NO: 6; to an epitope comprising a residue from the region of residues 25-35 of SEQ ID NO: 5, or the corresponding sequence of SEQ ID NO: 6; to an epitope comprising a residue from the region of residues 133-165 of SEQ ID NO: 5, or the corresponding sequence of SEQ ID NO: 6; to an epitope comprising a residue from the region of residues 268-276 of SEQ ID NO: 5, or the corresponding sequence of SEQ ID NO: 6; to an epitope comprising a residue from the region of residues 368-380 of SEQ ID NO: 5, or the corresponding sequence of SEQ ID NO: 6; and ii) the second binding domain binds to domain DI, DII, or Dill of the extracellular domain of CD5; to an epitope comprising residues from the region of residues 35-133 of SEQ ID NO: 5 (Domain DI), or the corresponding sequence of SEQ ID NO: 6; to an epitope comprising residues from the region of residues 165-268 of SEQ ID NO: 5 (Domain DII), or the corresponding sequence of SEQ ID NO: 6; to an epitope comprising residues from the region of residues 276-368 of SEQ ID NO: 5 (Domain Dill), or the corresponding sequence of SEQ ID NO: 6; to an epitope comprising a residue from the region of residues 25-35 of SEQ ID NO: 5, or the corresponding sequence of SEQ ID NO: 6; to an epitope comprising a residue from the region of residues 133-165 of SEQ ID NO: 5, or the corresponding sequence of SEQ ID NO: 6; to an epitope comprising a residue from the region of residues 268-276 of SEQ ID NO: 5, or the corresponding sequence of SEQ ID NO: 6; to an epitope comprising a residue from the region of residues 368-380 of SEQ ID NO: 5, or the corresponding sequence of SEQ ID NO: 6, wherein the first binding domain and second binding domain bind to the same domain, different epitopes of the same domain, or to different domains.

[0261] In some embodiments, the first binding domain and the second binding domain do not bind to the same domain of DI, DII, or Dill of CD5. In some embodiments, the first binding domain binds to DI, DII, Dill, the linker between DI and DII, the linker between DII and Dill, or the linker between Dill and the transmembrane domain of the CD5. In some embodiments,

[0262] the second binding domain binds to DI, DII, Dill, the linker between DI and DII, the linker between DII and Dill, or the linker between Dill and the transmembrane domain of the CD5.

[0263] -27- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT

[0264] In some embodiments, the first binding domain binds to domain DI and the second binding domain binds to domain Dill.

[0265] In some embodiments, the first binding domain binds to DI, DII, Dill, the linker between DI and DII, the linker between DII and Dill, or the linker between Dill and the transmembrane domain of the CD5; and the second binding domain binds to DI, DII, Dill, the linker between DI and DII, the linker between DII and Dill, or the linker between Dill and the transmembrane domain of the CD5, provided that the first and second binding domain do not bind to the same domain, linker, or the same epitope. In some embodiments, first binding domain binds to DI domain of CD5 and the second binding domain binds to Dill domain of CD5.

[0266] In some embodiments, the second binding domain binds to a Pan T-Cell surface protein. A “Pan T-Cell surface protein” refers to a T-cell protein that is expressed on the cell surface of the T cell and is ubiquitously expressed across more than one or most T-cell types. Examples of such Pan T-Cell surface proteins include, but are not limited to, PD-1, CD7, CD2, CD3, PD-1, CD-28, TCR, MHC, CD4, CD8, TIGIT, CD25, CCR5, CXCR4, CDla, CD28, CTLA-4, ICOS, 4-1BB (CD137), LFA-1, VLA-4, CD44, PSGL-1, CD6, CD40L, CD45RO, CD45RA, CD62L, CXCR3, CCR7, CD27, Fas (CD95), CD38, LAG-3, TIM-3, CD16, CD56, or CD107.

[0267] In some embodiments, the second binding domain binds to a recombinant engineered T cell specific target cell antigen. A “recombinant engineered T cell specific target cell antigen” is an antigen that is expressed on the outer surface of T cell and is not native to the T cell. An example of such an engineered antigen would be a chimeric antigen receptor that has been introduced into such T cell. These T cells can be present in a subject that can be treated with the polypeptides provided for herein. Other examples can be a linker present in a recombinant protein, such as a peptide linker. Thus, the polypeptides can be used to target antigens that are not normally present on the surface of a T-cell. Non-limiting examples of peptide linkers, include glycine / serine linkers and other examples of such linkers are provided for herein. Antigen binding domains can be made that specifically bind to such regions. In some embodiments, the second binding domain binds to an antigen binding domain of a chimeric antigen receptor present on the surface of an engineered T cell.

[0268] As provided for herein, the first binding domain can be an antibody or ligand that binds to CD5 and the second binding domain can be an antibody or ligand that binds to the immune cell surface protein, which can include CD5, or the recombinant engineered T cell specific target cell antigen. In some embodiments, as provided for herein, the first binding -28- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT

[0269] domain is a scFv antibody or a Fab antibody and the second binding domain is a scFv antibody or a Fab antibody. In some embodiments, the first binding domain is an scFv antibody and the second binding domain is an scFv antibody. In some embodiments, the first binding domain is an scFv antibody and the second binding domain is a Fab antibody. In some embodiments, the first binding domain is a Fab antibody and the second binding domain is a Fab antibody. In some embodiments, the first binding domain is a Fab antibody and the second binding domain is an scFv antibody. These are non-limiting configurations of such polypeptides and other configurations are provided for and described herein, and above, which includes those provided for in FIG. 1, FIG. 2, and Other formats for multi-specific antibodies can be found in U. S. Patent No. 11,739,144 and U. S. Application Publication No.

[0270] 20240052034, each of which is hereby incorporated by reference in its entirety.

[0271] In some embodiments, the polypeptide comprises a third binding domain (effector moiety) that binds to a cell specific antigen, such as a target cell or tumor antigen, or an immune cell surface antigen that is a different protein from what the first binding domain and the second binding domain binds to. In some embodiments, the target cell or tumor antigen is, but not limited to, CD 19, CD22, TAG-72, MUC16, PSMA, EGFR, a-integrin, BCMA, HER2, or Mesothelin, CLDN6, Nectin-4, CEA, and the like.

[0272] In some embodiments, the polypeptide comprises a third binding domain (effector moiety) that binds to a recombinant engineered T cell specific target cell antigen, provided that the second binding domain binds to an immune cell surface protein that is not the recombinant engineered T cell specific target cell antigen.

[0273] In some embodiments, the polypeptide comprises a first binding domain that binds to Domain DI of CD5 and the second binding domain binds to Domain DI, II or III of CD5. In some embodiments, the first binding domain is a Fab, scFv, or CD5 ligand. In some embodiments, the second binding domain is a Fab or scFv.

[0274] In some embodiments, the polypeptide comprises a first binding domain binds to Domain DI of CD5 and the second binding domain binds to a Pan T-Cell surface protein. In some embodiments, the first binding domain is a Fab, scFv, or CD5 ligand and the second binding domain is a Fab, scFv, or a ligand of the Pan T-Cell surface protein.

[0275] In some embodiments, the polypeptide comprises a first binding domain that binds to Domain DI or DI, II or III of CD5 and at least one additional binding domain (second or third binding domains) that binds to an extracellular domain of CD7 and / or PD-1 and / or other immunomodulatory T cell antigens, such as pro-inflammatory or anti-inflammatory signaling

[0276] -29- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT

[0277] pathways and cytokine / chemokine receptors, such as but not limited to, CD3, CD2, CD40L, CD28 / CTLA-4, 4-1BB, IL-18R, IL-12R, IL-10R, TGF0R, and the like. In some embodiments, the first binding domain is a Fab, scFv, or CD5 ligand and the second binding domain is a Fab, scFv, or a ligand of CD7 or PD-1 or of the immunomodulatory T cell antigen.

[0278] In some embodiments, the polypeptide comprises a first binding domain that binds to Domain DI of CD5, a second domain that binds to Domain DI, II or III of CD5 or to an extracellular domain of CD7, and a third binding domain that binds to a target cell antigen on a target cell, such as but not limited to CD 19, CD22, TAG-72, MUC16, PSMA, EGFR, a-integrin, BCMA, HER2, or Mesothelin, CLDN6, Nectin-4, CEA. In some embodiments, the first, second, and third binding domain can each be, independently, a Fab, ScFV, or ligand of the binding partner or target region of the protein.

[0279] In some embodiments, the polypeptide comprises a first binding domain that binds to Domain DI of CD5, a second binding domain that binds to a recombinant Engineered T cell specific target cell antigen, and optionally a third binding domain that binds to an extracellular domain of CD7 and / or PD1 or another pro-inflammatory T cell antigen. In some embodiments, the first binding domain, the second binding domain, and the third binding domain, can each be, independently, a Fab, scFv, or ligand of the binding partner of the binding domain.

[0280] Although some of the embodiments provided herein provide that the binding domain bind to DI, DII or Dill can also be substituted for the target of the binding domain if not already recited in the embodiment.

[0281] In some embodiments, the binding domain which binds to CD5 binds to the same epitope or a similar epitope as an anti-CD5 antibody having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 7 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 8.

[0282] QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLR WMGWINTHTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTAT YFCTRRGYDWYFDVWGAGTTVTVSS (SEQ ID NO: 7) DIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIY RANRLVDGVPSRFSGSGSGQDYSLTISSLDYEDMGIYYCQQYDESPWT FGGGTKLEIK (SEQ ID NO: 8).

[0283] -30- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT

[0284] In some embodiments, the binding domain which binds to CD5 binds to the same epitope or a similar epitope as an anti-CD5 scFv having the amino acid sequence of SEQ ID NO: 9.

[0285] MEFGLSWLFLVAILKGVQCIDAMGNIQLVQSGPELKKPGETVKISCKA SGYTFTNYGMNWVKQAPGKGLRWMGWINTHTGEPTYADDFKGRFA FSLETSASTAYLQINNLKNEDTATYFCTRRGYDWYFDVWGAGTTVTV SSGGGGSGGGGSGGGGSDIKMTQSPSSMYASLGERVTITCKASQDINS YLSWFHHKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLDY EDMGIYYCQQYDESPWTFGGGTKLEMKGSGDPA (SEQ ID NO: 9)

[0286] In some embodiments, the binding domain which binds to CD5 binds to the extracellular domain of CD5. In some embodiments, binding to the extracellular domain of CD5 leads to, or results in, internalization of CD5. In some embodiments, the binding domain which binds to CD5 binds to the extracellular domain of CD5 and leads to internalization of CD5. In some embodiments, binding to the extracellular domain of CD5 leads to, or results in, the degradation of the extracellular domain of CD5, the cytoplasmic domain of CD5, or the extracellular domain and the cytoplasmic domain of CD5. In some embodiments, the binding domain which binds to CD5 binds to the extracellular domain of CD5 and leads to the degradation of the extracellular domain of CD5, the cytoplasmic domain of CD5, or the extracellular domain and the cytoplasmic domain of CD5. In some embodiments, binding to the extracellular domain of CD5 leads to, or results in, the cleavage of the extracellular domain of CD5. In some embodiments, the binding domain which binds to CD5 binds to the extracellular domain of CD5 and leads to the cleavage of the extracellular domain of CD5.

[0287] In some embodiments, nucleic acid molecules are provided that encode for the polypeptides provided for herein. Due to the degenerate nature of the genetic code a variety of nucleic acid molecules can be used to encode for the same polypeptide. In some embodiments, the polypeptides are encoded by one nucleic acid molecule having different open reading frames and promoters operably connected to the same, or more than one nucleic acid molecule encoding for the polypeptides. The nucleic acid molecules can then be used to produce the polypeptides for which they encode.

[0288] For example, the nucleic acid molecules can be transduced or transfected into a cell, such as a eukaryotic cell, or human cell, that is then cultured under suitable conditions to produce the polypeptides. Methods of making such polypeptides are routine and any method can be used by one of skill in the art.

[0289] -31- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT

[0290] Thus, in some embodiments, a host cell is provided that comprises the nucleic acid molecule or molecules encoding the polypeptides. The host cell can be a eukaryotic cell. In some embodiments, the host cell is a human derived cell. In some embodiments, the eukaryotic cell is a mammalian cell, such as a CHO cell. In some embodiments, the cell is a yeast cell. In some embodiments, the cell is a bacterial cell.

[0291] In some embodiments, methods of producing polypeptides provided herein are provided. In some embodiments, the methods comprise culturing a host cell comprising one or more nucleic acid molecules encoding the polypeptide under conditions to express and produce the polypeptide. In some embodiments, the method further comprises isolating the expressed polypeptide.

[0292] The polypeptides provided for herein can be formulated or be part of compositions, such as pharmaceutical compositions. Accordingly, in some embodiments, the present embodiments provide compositions, e.g., pharmaceutically acceptable compositions, which comprise a polypeptide provided for herein. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable excipient or carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, excipients, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible. The carrier can be suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, local, ophthalmic, topical, spinal or epidermal administration (e.g. by injection or infusion). In some embodiments, pharmaceutical carriers can also be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The pharmaceutical carriers can also be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary, stabilizing, thickening, lubricating and coloring agents can be used. The carriers can be used in pharmaceutical compositions comprising the polypeptides provided for herein.

[0293] The compositions and compounds of the embodiments provided for herein may be in a variety of forms. These include, for example, liquid, lyophilized, and other dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions. The form depends on the intended mode of administration and therapeutic application. Typical compositions are in the form of injectable or infusible solutions. In some embodiments, the mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In some embodiments, the composition is administered by intravenous infusion or injection. In some embodiments, the composition is administered by -32- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT

[0294] intramuscular or subcutaneous injection. In some embodiments, the composition is administered locally, e.g., by injection, or topical application, to a target site.

[0295] The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrastemal injection and infusion.

[0296] The compositions typically should be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high therapeutic molecule concentration. Sterile injectable solutions can be prepared by incorporating the active compound (i.e., therapeutic molecule) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

[0297] As will be appreciated by the skilled artisan, the route and / or mode of administration will vary depending upon the desired results. In certain embodiments, the active compound may be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, poly orthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0298] -33- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT

[0299] The compositions can also be administered with medical devices known in the art, such as an auto-injector.

[0300] Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0301] An exemplary, non-limiting range for a therapeutically or prophylactically effective amount of polypeptide is 0.1-30 mg / kg, more preferably 1-25 mg / kg. Dosages and therapeutic regimens of the therapeutic compound can be determined by a skilled artisan. In certain embodiments, the therapeutic compound is administered by injection (e.g., subcutaneously or intravenously) at a dose of about 1 to 40 mg / kg, e.g., 1 to 30 mg / kg, e.g., about 5 to 25 mg / kg, about 10 to 20 mg / kg, about 1 to 5 mg / kg, 1 to 10 mg / kg, 5 to 15 mg / kg, 10 to 20 mg / kg, 15 to 25 mg / kg, or about 3 mg / kg. The dosing schedule can vary from e.g., once a week to once every 2, 3, or 4 weeks. In one embodiment, the therapeutic compound is administered at a dose from about 10 to 20 mg / kg every other week. The therapeutic compound can be administered by intravenous infusion at a rate of more than 20 mg / min, e.g., 20-40 mg / min, and typically greater than or equal to 40 mg / min to reach a dose of about 35 to 440 mg / m2, typically about 70 to 310 mg / m2, and more typically, about 110 to 130 mg / m2. In embodiments, the infusion rate of about 110 to 130 mg / m2 achieves a level of about 3 mg / kg. In other embodiments, the therapeutic compound can be administered by intravenous infusion at a rate of less than 10 mg / min, e.g., less than or equal to 5 mg / min to reach a dose of about 1 to 100 mg / m2, e.g., about 5 to 50 mg / m2, about 7 to 25 mg / m2, or, about 10 mg / m2. In some embodiments, the polypeptide is infused over a period of about 30 min. It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.

[0302] -34- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT

[0303] The pharmaceutical compositions may include a "therapeutically effective amount" of the polypeptide. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of a therapeutic molecule may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the therapeutic compound to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of a therapeutic molecule t is outweighed by the therapeutically beneficial effects. A "therapeutically effective dosage" preferably inhibits a measurable parameter, e.g., cell proliferation or growth at least about 20%, by at least about 40%, by at least about 60%, or by at least about 80% relative to untreated subjects. The ability of a compound to inhibit a measurable parameter, e.g., cell growth or proliferation, can be evaluated in an animal model system predictive of efficacy in cancer. Alternatively, this property of a composition can be evaluated by examining the ability of the compound to inhibit, such inhibition in vitro by assays known to the skilled practitioner.

[0304] Also within the scope of the embodiments is a kit comprising the polypeptides provided for herein. The kit can include one or more other elements including: instructions for use; other reagents, e.g., a label, a pharmaceutical composition, devices or other materials for preparing the pharmaceutical composition for administration; pharmaceutically acceptable carriers; and devices or other materials for administration to a subject.

[0305] As provided for herein, the polypeptides can be used in conjunction with CAR-T therapy.

[0306] A chimeric antigen receptor (CAR) comprises an antigen binding domain, a transmembrane domain, and an intracellular domain. The “Antigen Binding Domain” refers to the domain of the CAR that binds to a specific antigen, such as a tumor antigen. Accordingly, in some embodiments, the CAR comprises a target-specific binding element otherwise referred to as an antigen binding domain. The choice of antigen binding domain depends upon the type and number of ligands that define the surface of a target cell. For example, the antigen binding domain may be chosen to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state. In some embodiments, the CAR can be engineered to target a tumor antigen. The antigens discussed herein are merely included by way of example. The list is not intended to be exclusive and further examples will be readily apparent to those of skill in the art. Tumor antigens are proteins that are produced by tumor cells that elicit an immune response, such as, but not limited to, T-cell mediated immune responses. The selection of the antigen binding domain of can depend on the particular type -35- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT

[0307] of cancer or disease to be treated. Tumor antigens are well known in the art and include, for example, a glioma-associated antigen, carcinoembryonic antigen (CEA), P-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA, Her2 / neu, survivin and telomerase, prostate-carcinoma tumor antigen- 1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, BCMA, PSMA, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor and mesothelin.

[0308] The type of tumor antigen referred may also be a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA). A TSA is unique to tumor cells and does not occur on other cells in the body. A TAA associated antigen is not unique to a tumor cell and instead is also expressed on a normal cell under conditions that fail to induce a state of immunologic tolerance to the antigen. The expression of the antigen on the tumor may occur under conditions that enable the immune system to respond to the antigen. TAAs may be antigens that are expressed on normal cells during fetal development when the immune system is immature and unable to respond or they may be antigens that are normally present at extremely low levels on normal cells but which are expressed at much higher levels on tumor cells.

[0309] Non-limiting examples of TSA or TAA antigens include the following:

[0310] Differentiation antigens such as MART-l / MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2 and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor-suppressor genes such as p53, Ras, HER-2 / neu; unique tumor antigens resulting from chromosomal translocations; such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as the Epstein Barr virus antigens EBVA and the human papillomavirus (HPV) antigens E6 and E7. Other large, protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl85erbB2, pl80erbB-3, c-met, nm-23Hl, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-Catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, M0V18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-associated protein, TAAL6, TAG72, TLP, and TPS.

[0311] -36- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT

[0312] With respect to the transmembrane domain of a CAR, the CAR can be designed to comprise a transmembrane domain that is fused to the extracellular domain of the CAR. In some embodiments, the transmembrane domain that naturally is associated with one of the domains in the CAR is used. In some embodiments, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

[0313] In some embodiments, the transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. Transmembrane regions of particular use in this invention may be derived from (i.e. comprise at least the transmembrane region(s) 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, CD154. Alternatively the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. In some embodiments, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. Optionally, a short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length may form the linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR. A glycine-serine doublet can also be used as a suitable linker.

[0314] The intracellular domain or otherwise the cytoplasmic domain of the CAR is responsible for activation of at least one of the normal effector functions of the immune cell in which the CAR has been placed in. 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. Thus the term “intracellular domain” refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire intracellular 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 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 domain is thus meant to include any truncated portion of the intracellular domain sufficient to transduce the effector function signal.

[0315] Non-limiting examples of intracellular domains for use in a CAR include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to -37- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT

[0316] initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any synthetic sequence that has the same functional capability.

[0317] It is known that signals generated through the TCR alone are insufficient for full activation of the T cell and that a secondary or co-stimulatory signal is also required. Thus, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequence: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences) and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences).

[0318] Primary cytoplasmic signaling sequences regulate primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs.

[0319] Examples of ITAM containing primary cytoplasmic signaling sequences that are of particular use in the invention include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. It is particularly preferred that cytoplasmic signaling molecule in the CAR of the invention comprises a cytoplasmic signaling sequence derived from CD3 zeta.

[0320] In some embodiments, the intracellular domain of the CAR can be designed to comprise the CD3-zeta signaling domain by itself or combined with any other desired intracellular domain(s) useful in the context of the CAR of the invention. For example, the intracellular domain of the CAR can comprise a CD3 zeta chain portion and a costimulatory signaling region. The costimulatory signaling region refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or their ligands that is required for an efficient response of lymphocytes to an antigen. Examples of such molecules include, but are not limited to, CD27, CD28, 4-1BB (CD137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen- 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83, and the like.

[0321] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of a CAR may be linked to each other in a random or specified order. Optionally, a short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length may form the linkage. A glycine-serine doublet can also be used as a suitable linker.

[0322] -38- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT

[0323] In some embodiments, the intracellular domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of CD28. In another embodiment, the intracellular domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of 4-1BB. In yet another embodiment, the intracellular domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of CD28 and 4-1BB.

[0324] These embodiments of different CARs are for exemplary purposes only and the polypeptides that bind to CD5 and at least one other surface molecule on an immune cell can be used in conjunction with any CAR.

[0325] Cells

[0326] In some embodiments, a cell is provided. In some embodiments, the cell is a T-cell. In some embodiments, the T-cell is engineered to express a chimeric antigen receptor and a polypeptide on the surface of the T-cell, wherein the polypeptide comprises an antigen binding domain. In some embodiments, the antigen binding domain of the polypeptide binds to a target antigen as provided for herein. In some embodiments, the antigen binding domain of the polypeptide binds to a protein expressed on the surface of the T-cell. In some embodiments, the antigen binding domain of the polypeptide binds to CD5.

[0327] In some embodiments, the polypeptide that binds to CD5 is as provided for herein. In some embodiments, the polypeptide that binds to CD5 binds to a region in CD5 as provided for herein. In some embodiments, the polypeptide that binds to CD5 binds to the same epitope as an anti-CD5 antibody, or antigen binding fragment thereof, comprising a variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 7 and a variable light chain domain comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the the polypeptide that binds to CD5 binds to the same epitope as an anti-CD5 scFv comprising the amino acid sequence of SEQ ID NO: 9.

[0328] In some embodiments, the polypeptide that binds to CD5 binds to the extracellular domain of CD5. In some embodiments, the binding to the extracellular domain of CD5 results in internalization of the CD5. In some embodiments, the binding to the extracellular domain of CD5 results in the degradation of the extracellular domain of CD5, the cytoplasmic domain of CD5, or both the extracellular domain of CD5 and the cytoplasmic domain of CD5. In some embodiments, the binding to the extracellular domain of CD5 results in cleavage of the extracellular domain of CD5. In some embodiments, the polypeptide that binds to CD5 leads to internalization of CD5, thus reducing the amount of CD5 on the surface of the cell. In some embodiments, the polypeptide that binds to CD5 -39- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT

[0329] results in the degradation of CD5. The degradation can be, for example, due to the internalization and processing of the protein internally, such as through a lysosome or other degradation pathway. In some embodiments, the degradation is due to the CD5 protein on the surface of the cell be cleaved, e.g., proteolytic cleavage. In some embodiments, the polypeptide that binds to CD5 is a protac or lytac molecule or any other molecule that can bind to and degrade CD5. The protac or lytac can, in some embodiments, lead to internalization and degradation, but it can also lead to degradation (proteolysis) through other pathways. In some embodiments, the polypeptide that binds to CD5 is an antibody that binds to CD5. The antibody can be a monovalent antibody, in that it only has one antigen domain that bind to one epitope. In some embodiments, the antibody is a biparatopic antibody, meaning that the antibody comprises at least two antigen binding domains that bind to different epitopes on CD5. A biparatopic antibody can be referred to as a bispecific antibody, even if the different antigen binding domains bind to the same antigen (e.g. CD5), but bind to different epitopes. The different epitopes can be, in some embodiments, overlapping epitopes, but not identical, or non-overlapping epitopes.

[0330] In some embodiments, the T-cell comprises a chimeric antigen receptor and an antibody, such as but not limited to, an scFv, on the surface of the T-cell, wherein the antibody (e.g. scFv) binds to CD5. In some embodiments, the antibody (e.g. scFv) that binds to CD5 is as provided for herein. In some embodiments, the antibody that binds to CD5 binds to a region in CD5 as provided for herein. In some embodiments, the antibody that binds to CD5 binds to the same epitope as an anti-CD5 antibody, or antigen binding fragment thereof, comprising a variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 7 and a variable light chain domain comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the antibody that binds to CD5 binds to the same epitope as an anti-CD5 scFv comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the antibody that binds to CD5 binds to the extracellular domain of CD5. In some embodiments, the binding to the extracellular domain of CD5 results in internalization of the CD5. In some embodiments, the binding to the extracellular domain of CD5 results in the degradation of the extracellular domain of CD5, the cytoplasmic domain of CD5, or both the extracellular domain of CD5 and the cytoplasmic domain of CD5. In some embodiments, the binding to the extracellular domain of CD5 results in cleavage of the extracellular domain of CD5. In some embodiments, the chimeric antigen receptor comprises an antigen binding domain that does not bind to CD5. In some embodiments, the chimeric antigen receptor comprises an antigen binding domain that binds to CD5.

[0331] -40- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT

[0332] In some embodiments, the T-cell as provided for herein expresses CD5.

[0333] Methods of Use

[0334] The compounds provided for herein can be used to treat cancers in a subject. In some embodiments, the subject is a subject “in need thereof.” In some embodiments, the cancer is T-cell lymphoma or a T cell leukemia. In some embodiments, the cancer is acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL). In some embodiments, the cancer is glioblastoma multiforme (GBM), anaplastic astrocytoma, giant cell glioblastoma, gliosarcoma, anaplastic oligodendroglioma, anaplastic ependymoma, choroid plexus carcinoma, anaplastic ganglioglioma, pineoblastoma, medulloepithelioma, ependymoblastoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, atypical teratoid / rhabdoid tumor, lung cancer (e.g., non-small cell lung carcinomas) breast, prostate, ovarian, colorectal and bladder carcinoma and any combination thereof, and metastases of any of the cancers. In some embodiments, the cancer is acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vagina, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor, glioma, Hodgkin lymphoma, hypopharynx cancer, kidney cancer, larynx cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma, cancer of the oropharynx, ovarian cancer, cancer of the penis, pancreatic cancer, peritoneum, omentum, and mesentery cancer, pharynx cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, cancer of the uterus, ureter cancer, and urinary bladder cancer.

[0335] Accordingly, in some embodiments, methods of treating cancer in a subject are provided. In some embodiments, the methods comprise administering to the subject a pharmaceutical composition comprising one or more polypeptides as provided for herein. In some embodiments, the subject is a subject in need thereof. In some embodiments, the subject has previously been treated with CAR-T therapy. In some embodiments, the CAR-T therapy is ex-vivo CAR-T therapy or in-vivo CAR-T therapy. “Ex-vivo CAR-T therapy”

[0336] -41- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT

[0337] refers to when the CAR-T cells are produced outside of the subject and then are administered to the subject. “In-vivo CAR-T therapy’’ refers to a therapeutic when the CAR-T cells are produced in vivo by administering a delivery therapeutic to the subject that infects or transduces T Cells to produce the CAR-T cells. A non-limiting examples of such “in vivo CAR-T” therapy is provided for in PCT Publication Nos. WO2023114884 and WO2024026284, each of which is hereby incorporated by reference in its entirety. Without being bound to any particular theory, CAR-T cells once generated may have their activity be reduced due to possible negative feedback loops present in vivo. The polypeptides can be administered to such patients or along with the CAR-T therapy to enhance or re-activate the CAR-T therapy. Accordingly, in some embodiments, methods of enhancing, re-activating, or others wise agonizing CAR-T therapy are provided by administering a pharmaceutical composition comprising a polypeptide provided for herein to a subject who has been previously treated with CAR-T therapy.

[0338] In some embodiments, the subject has not been previously treated with CAR-T therapy.

[0339] In some embodiments, the methods comprise co-administering the polypeptide and an additional cancer therapeutic. In some embodiments, the additional cell therapeutic is an engineered cell therapy or gene therapy to treat the cancer. In some embodiments, the engineered cell therapy is CAR-T cell therapy, including but not limited to ex-vivo CAR-T cell therapy or in-vivo CAR-T cell therapy. The therapeutics can be administered simultaneously or sequentially. If the polypeptide is administered after the patient has been treated with CAR-T cell therapy, the polypeptide can be administered the following day, one week, two weeks, three weeks, four weeks, eight weeks, 12 weeks, 16 weeks, or longer after being treated with the CAR-T therapy.

[0340] In some embodiments, the subject has a cancer that is not a CD5 positive cancer. In some embodiments, the cancer is a cancer that is not a T-cell lymphoma.

[0341] The contacting or administration of the polypeptide can occur by any method, such as those provided for herein, including, but not limited to intravenous or subcutaneous administrations.

[0342] In some embodiments, methods of reducing CD5 surface expression on an immune cell are provided. In some embodiments, the methods comprising contacting an immune cell expressing CD5 on its surface with a pharmaceutical composition comprising a polypeptide -42- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT

[0343] as provided for herein. In some embodiments, the immune cell is a CAR-T cell. In some embodiments, the methods comprise administering to a subject comprising the immune cell the pharmaceutical composition. As provided herein, without being bound to any particular theory, the ability to reduce the surface expression of CD5 can lead to propagating pro-inflammatory / effector signal into the T cell. Alternatively, blocking the internalization of CD5 with a polypeptide can down-regulate immune cell effector function. Thus, in some embodiments, the methods provided for herein can be used to activate the cells or suppress effector function by contacting the cells with the compositions and polypeptides provided for herein.

[0344] ENUMERATED EMBODIMENTS

[0345] In some embodiments, the following embodiments are provided:

[0346] 1. A polypeptide comprising a first binding domain (effector moiety) and a second binding domain (effector moiety), wherein:

[0347] the first binding domain binds to an extracellular domain of CD5: and the second binding domain binds to an immune cell surface protein.

[0348] 2. The polypeptide of embodiment 1, wherein the second binding domain binds to an extracellular domain of CD5.

[0349] 3. The polypeptide of embodiment 2, wherein the first binding domain and the second binding domain bind to the same or different CD5 epitopes.

[0350] 4. The polypeptide of embodiment 2, wherein the first binding domain and the second binding domain bind to the same CD5 epitope.

[0351] 5. The polypeptide of any one of embodiments 1-4, wherein the first binding domain binds to the DI domain, the DII domain, or the Dill domain of the extracellular domain of CD5 and the second binding domain binds to the DI domain, the DII domain, or the Dill domain of the extracellular domain of CD5, wherein the first binding domain and second binding domain bind to the same or different domains.

[0352] 6. The polypeptide of embodiment 5, wherein the first binding domain and the second binding domain do not bind to the same domain of DI, DII, or Dill of CD5.

[0353] 7. The polypeptide of any one of embodiments 1-6, wherein the first binding domain binds to DI, DII, Dill, the linker between DI and DII, the linker between DII and Dill, or the linker between Dill and the transmembrane domain of the CD5.

[0354] -43- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT

[0355] 8. The polypeptide of any one of embodiments 1-7, wherein the second binding domain binds to DI, DII, Dill, the linker between DI and DII, the linker between DII and Dill, or the linker between Dill and the transmembrane domain of the CD5.

[0356] 9. The polypeptide of embodiment 1, wherein:

[0357] the first binding domain binds to DI, DII, Dill, the linker between DI and DII, the linker between DII and Dill, or the linker between Dill and the transmembrane domain of the CD5: and

[0358] the second binding domain binds to DI, DII, Dill, the linker between DI and DII, the linker between DII and Dill, or the linker between Dill and the transmembrane domain of the CD5,

[0359] provided that the first and second binding domains do not bind to the same domain, linker, or the same epitope.

[0360] 10. The polypeptide of any one of embodiments 1-9, wherein the first binding domain binds to DI domain of CD5 and the second binding domain binds to Dill domain of CD5. 11. The polypeptide of any one of embodiments 1-10, wherein the first binding domain when bound to CD5 leads to its internalization or degradation of CD5.

[0361] 12. The polypeptide of embodiment 1, wherein the second binding domain binds to a Pan T-Cell surface protein.

[0362] 13. The polypeptide of embodiment 12, wherein the Pan T-Cell surface protein is PD-1, CD7, CD2, CD3, PD-1, CD-28, TCR, MHC, CD4, CD8, TIGIT, CD25, CCR5, CXCR4, CD la, CD28, CTLA-4, ICOS, 4-1BB (CD137), LFA-1, VLA-4, CD44, PSGL-1, CD6, CD40L, CD45RO, CD45RA, CD62L, CXCR3, CCR7, CD27, Fas (CD95), CD38, LAG-3, TIM-3, CD16, CD56, or CD107.

[0363] 14. The polypeptide of embodiment 13, wherein the Pan T-Cell surface protein is CD7 or CD3.

[0364] 15. The polypeptide of any one of embodiments 1-11, wherein the second binding domain binds to a recombinant engineered T cell specific target cell antigen.

[0365] 16. The polypeptide of embodiment 15, wherein the recombinant engineered T cell specific target cell antigen is peptide linker (e.g., glycine / serine, and the like) or directed to a non-native antibody sequence present on the surface of the T cell, such as, but not limited to the antigen binding domain of a chimeric antigen receptor present on the surface of an engineered T cell.

[0366] 17. The polypeptide of any one of embodiments 1-16, wherein the first binding domain is an antibody or ligand that binds to CD5 and the second binding domain is an antibody or -44- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT

[0367] ligand that binds to the immune cell surface protein or the recombinant engineered T cell specific target cell antigen.

[0368] 18. The polypeptide of embodiment 17, wherein the first binding domain is a scFv antibody or a Fab antibody and the second binding domain is a scFv antibody or a Fab antibody.

[0369] 19. The polypeptide of embodiment 17, wherein the first binding domain is an scFv antibody and the second binding domain is an scFv antibody.

[0370] 20. The polypeptide of embodiment 17, wherein the first binding domain is an scFv antibody and the second binding domain is a Fab antibody.

[0371] 21. The polypeptide of embodiment 17, wherein the first binding domain is a Fab antibody and the second binding domain is a Fab antibody.

[0372] 22. The polypeptide of embodiment 17, wherein the first binding domain is a Fab antibody and the second binding domain is an scFv antibody.

[0373] 23. The polypeptide of any one of embodiments 1-22, wherein the polypeptide comprises a third binding domain (effector moiety) that binds to a cell specific antigen, such as a target cell or tumor antigen, or an immune cell surface antigen that is a different protein from what the first binding domain and the second binding domain binds to.

[0374] 24. The polypeptide of embodiment 23, wherein the target cell antigen is CD19, CD22, TAG-72, MUC16, PSMA, EGFR, a-integrin, BCM A, HER2, Mesothelin, CLDN6, Nectin-4, CEA, and the like.

[0375] 25. The polypeptide of any one of embodiments 1-22, wherein the polypeptide comprises a third binding domain (effector moiety) that binds to a recombinant engineered T cell specific target cell antigen, provided that the second binding domain binds to an immune cell surface protein that is not the recombinant engineered T cell specific target cell antigen. 26. The polypeptide of embodiment 25, wherein the recombinant engineered T cell specific target cell antigen is peptide linker (e.g., glycine / serine, and the like) or directed to a non-native antibody sequence present on the surface of the T cell, such as, but not limited to the antigen binding domain of a chimeric antigen receptor present on the surface of an engineered T cell.

[0376] 27. A pharmaceutical composition comprising the polypeptide of any one of embodiments 1-26 and a pharmaceutically acceptable excipient.

[0377] 28. A nucleic acid molecule or molecules encoding the polypeptide of any one of embodiments 1-26.

[0378] -45- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT

[0379] 29. A host cell comprising the nucleic acid molecule or molecules of embodiment 28. 30. A method of producing the polypeptide of any one of embodiments 1-26, the method comprising culturing a host cell comprising one or more nucleic acid molecules encoding the polypeptide under conditions to express and produce the polypeptide.

[0380] 31. The method of embodiment 30, wherein the method further comprises isolating the expressed polypeptide.

[0381] 32. A method of treating cancer or an auto-immune disease in a subject, the method comprising administering to the subject a pharmaceutical composition comprising the polypeptide of any one of embodiments 1-26.

[0382] 33. The method of embodiment 32, wherein the subject has previously been treated with CAR-T therapy.

[0383] 34. The method of embodiment 33, wherein the CAR-T therapy is ex-vivo CAR-T therapy or in-vivo CAR-T therapy.

[0384] 35. The method of embodiment 32, wherein the subject has not been previously treated with CAR-T therapy.

[0385] 36. The method of embodiment 32, wherein the method comprises co-administering the polypeptide and an additional cancer therapeutic.

[0386] 37. The method of embodiment 36, wherein the additional cell therapeutic is an engineered cell therapy or gene therapy to treat the cancer.

[0387] 38. The method of embodiment 37, wherein the engineered cell therapy is CAR-T cell therapy.

[0388] 39. The method of embodiment 38, wherein the CAR-T cell therapy is ex-vivo CAR-T cell therapy or in-vivo CAR-T cell therapy.

[0389] 40. The method of any one of embodiments 32-39, wherein the cancer is not a CD5 positive cancer.

[0390] 41. The method of embodiment 40, wherein the cancer is not T-cell lymphoma.

[0391] 42. A method of reducing CD5 surface expression on an immune cell, the method comprising contacting the immune cell expressing CD5 on its surface with a pharmaceutical composition comprising the polypeptide of any one of embodiments 1-26.

[0392] 43. The method of embodiment 42, wherein the immune cell is a CAR-T cell.

[0393] 44. The method of embodiment 42 or 43, wherein the method comprises administering to a subject comprising the immune cell the pharmaceutical composition.

[0394] -46- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT

[0395] 45. A method of activating a T-cell, the method comprising contacting a T cell expressing CD5 on its surface with a pharmaceutical composition comprising the polypeptide of any one of embodiments 1-26.

[0396] 46. The method of embodiment 45, wherein the immune cell is a CAR-T cell.

[0397] 47. The method of embodiment 45 or 46, wherein the method comprises administering to a subject comprising the T cell the pharmaceutical composition.

[0398] 48. A T-cell comprising a chimeric antigen receptor and a polypeptide expressed on the surface of the T-cell, wherein the polypeptide expressed on the surface of the T-cell comprises an antigen binding domain that binds to a surface protein of the T-cell.

[0399] 49. The T-cell of embodiment 48, wherein the antigen binding domain binds to CD5. 50. The T-cell of embodiment 49, wherein the antigen binding domain that binds to CD5 comprises an scFv.

[0400] 51. The T-cell of embodiment 49 or 50, wherein the antigen binding domain that binds to CD5 binds to the same epitope as an anti-CD5 antibody, or antigen binding fragment thereof, comprising a variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 7 and a variable light chain domain comprising the amino acid sequence of SEQ ID NO: 8.

[0401] 52. The T-cell of any one of embodiments 49-51, wherein the antigen binding domain that binds to CD5 binds to the same epitope as an anti-CD5 scFv comprising the amino acid sequence of SEQ ID NO: 9.

[0402] 53. The T-cell of any one of embodiments 49-52, wherein the antigen binding domain that binds to CD5 binds to the extracellular domain of CD5.

[0403] 54. The T-cell of embodiment 53, wherein the binding to the extracellular domain of CD5 results in internalization of the CD5.

[0404] 55. The T-cell of embodiment 53, wherein the binding to the extracellular domain of CD5 results in degradation of the extracellular domain of CD5, the cytoplasmic domain of CD5, or both the extracellular domain of CD5 and the cytoplasmic domain of CD5.

[0405] 56. The T-cell of embodiment 53, wherein the binding to the extracellular domain of CD5 leads to the cleavage of the extracellular domain of CD5.

[0406] 57. The T-cell of any one of embodiments 48-56 wherein the T-cell is a CD5 positive T-cell.

[0407] 58. The T-cell of any one of embodiments 48-57, wherein the chimeric antigen receptor comprises an antigen binding domain that binds to CD5.

[0408] -47- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT

[0409] 59. The T-cell of any one of embodiments 48-57, wherein the chimeric antigen receptor comprises an antigen binding domain that does not bind to CD5.

[0410] EXAMPLES

[0411] The following examples are illustrative, but not limiting, of the compounds, compositions and methods described herein. Other suitable modifications and adaptations known to those skilled in the art are within the scope of the following embodiments.

[0412] Example 1: Tethered, But Not Soluble, Anti-CD5 Antibodies, Enhance Effector Function of CART Cells With Surface CD5 Expression. Plates were either left untreated or coated overnight at 4°C with the following antibodies (0.25 µg / mL): mIgG1 (Invitrogen, #02-6100), CD5 AB2, or CD5 AB3. The coating of the plates with the anti-CD5 antibodies, which tethers them to the plates mimics the effect of a bispecific antibody that can link a CD5 to more than one antigen, even if not both CD5, and allow the clustering of the CD5 receptor. After washing away unbound antibodies in treated wells, primary anti-CD19 CAR-T cells (CART19) were added and incubated overnight. Soluble CD5 antibodies, as indicated, were added to the untreated wells and incubated under the same conditions. Supernatants were collected from each condition and analyzed by ELISA to measure IFNy secretion, which is a measure of effector function of the CAR-T cells. IFNy levels are reported in pg / mL, calculated from a standard curve. As illustrated in FIG. 4A - 4C, the cells that were exposed to plates that were coated with anti-CD5 antibodies, as opposed to soluble anti-CD5 antibodies, had a significant increase IFNy secretion. This was demonstrated with two different anti-CD5 antibodies as shown. The results are shown to be CD5 specific, because the same assays were performed on CD5KO or a mock treated cells expressing the same CAR construct, and no significant increase in IFNy secretion was observed. Accordingly, tethering an anti-CD5 antibody, which would mimic a bispecific antibody comprising an anti-CD5 antibody can be used to reduce CD5 surface expression as discussed below, which, without being bound to any particular theory, leads to a pro-inflammatory / effector signal into the T cell and an activation signal being propagated internally and, therefore, the increase in IFNy secretion.

[0413] Example 2: Tethered Anti-CD5 And Anti-CD7 Antibodies Enhance Effector Function At Varying Levels Compared To Several Controls. The effect of the anti-CD5 antibodies -48- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT

[0414] illustrated in Example 1 was tested for anti-CD7 antibodies that were also tethered to a plate similarly to how the antibodies were tethered to the plates in Example 1. Briefly, plates were coated overnight at 4°C with the following antibodies (0.25 µg / mL): mIgG1 (Invitrogen, #02-6100), mIgG2a (Novus, #NBP1-96981), MHCI (HLA-ABC, Invitrogen, #14-9983-82), CD5 AB2, CD5 AB3, CD5 AB4, and CD7 AB1. After washing unbound antibodies, primary anti-CD19 T (CART 19) cells were added to each well and incubated overnight. Supernatants were then collected from each condition and analyzed by ELISA to measure IFNy secretion. IFNy levels are presented in pg / mL, calculated using a standard curve. As illustrated in FIG. 5, just like anti-CD5 antibodies, anti-CD7 antibodies that bind to the T-cell surface protein CD7, enhance the IFNy secretion. The same effect was not seen with other antibodies that simply bind to IgGl, IgG2 or MHCI. Thus, demonstrating the specific effects of the anti-CD5 and anti-CD7 antibodies, which can be utilized in a multi-specific format, such as a bispecific format, to enhance the activity of CAR-T cells.

[0415] Example 3: Tethered anti-CD5 and anti-CD7 antibodies synergize the effect of CAR-T Cells. To better illustrate the synergy that can be seen of a bispecific antibody comprising an anti-CD5 antigen binding domain and an anti-CD7 binding domain, plates were coated with either control antibodies, the CD5 antibody on its own, the CD7 antibody on its own or a combination of the CD5 and CD7 antibodies. Briefly, Plates were coated overnight at 4°C with the following antibodies (total per well = 0.25 µg / mL unless otherwise noted): MHCI (HLA-ABC, Invitrogen, #14-9983-82), RetroNectin (Takara, #T100A, 100 µg / mL), CD5 AB2, and CD7 AB1. After washing unbound antibodies, primary anti-CD19 T (CART19) cells were added to each well and incubated overnight. Supernatants were then collected from each condition and analyzed by ELISA to measure IFNy secretion. IFNy levels are presented in pg / mL, calculated using a standard curve. Data are shown as the mean ± standard error from duplicate wells. As illustrated in FIG. 6, in this experiment, although both the anti-CD5 antibody (CD5 AB2) and the anti-CD7 antibody (CD7 AB1) increased IFNy secretion over the control antibodies, the combination of the anti-CD5 and anti-CD7 antibodies provided a more significant increase, which was synergistic as compared to just the CD5 and CD7 antibodies alone. Thus, these results also illustrate the ability of such antibodies to enhance the effects of CAR-T cells and would be expected to do so in a bispecific format that allows the clustering of CD5 and / or CD7, which as discussed herein may be due to the reduction in CD5 surface expression, which leads to a pro-inflammatory / effector signal into the T cell.

[0416] -49- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT

[0417] Example 4: Tethered Anti-CD5 and Anti-CD7 Antibodies Synergize with Antigen Stimulation. We next tested to determine whether tethered anti-CD5 and anti-CD7 antibodies, either alone or in combination, could enhance the activity of CAR-T cells that had been exposed to the tumor antigen, which in this experiment was CD 19. Briefly, plates were coated overnight at 4°C with the following antibodies (total per well = 0.25 µg / mL): mIgG1 (Invitrogen, #02-6100), MHCI (HLA-ABC, Invitrogen, #14-9983-82), CD5 AB2, and CD7 AB1. After washing unbound antibodies, primary anti-CD19 T (CART19) cells, which had been stimulated by exposure to CD 19, were added to each well and incubated overnight. Supernatants were then collected from each condition and analyzed by ELISA to measure IFNy secretion. IFNy levels are presented in pg / mL, calculated using a standard curve. Data are shown as the mean ± standard error from duplicate wells. As illustrated in FIG. 7, both anti-CD5 and anti-CD7 antibodies on their own were able to induce IFNy secretion above control levels, which indicates that a tethered antibody, which mimics a bispecific antibody, can also enhance the IFNy secretion induced by an already antigen stimulated CAR-T cell. Additionally, it was found that the combination of anti-CD5 and anti-CD7 antibodies together was able to synergistically increase the IFNy secretion of such cells. Thus, these results also illustrate the ability of such antibodies to enhance the effects of CAR-T cells and would be expected to do so in a bispecific format that leads, as discussed herein can lead to reduced CD5 surface expression and, without being bound to any theory, an increase in IFNy secretion.

[0418] Example 5: Epitope specific Anti-CD5 antibodies induce surface CD5 downregulation compared to control antibodies. To help elucidate a possible mechanism of action for the activation of the CAR-T cells, we next measured the presence of CD5 on the surface of CAR-T cells after being exposed to tethered anti-CD5 antibodies or control antibodies that bind to CD7, MHC1, or IgGl. Briefly, plates were coated overnight at 4°C with the following antibodies (total per well = 0.25 µg / mL): mIgG1 (Invitrogen, #02-6100), MHCI (HLA-ABC, Invitrogen, #14-9983-82), CD5 AB2, and CD7 AB1. After washing unbound antibodies, primary anti-CD19 T (CART19) cells were added to each well and incubated overnight. Cells were then stained with UCHT2-APC (1:100; anti-CD5, BioLegend, #300612) or BLla-PacificBlue (1:50; anti-CD5, Beckman Coulter, #A82790), and CD5 surface expression was analyzed by flow cytometry. As illustrated in FIG. 8, the tethered anti-CD5 antibody induced surface CD5 downregulation, whereas the other control antibodies had no effect on CD5 surface expression. Without being bound to any particular theory, the internalization of CD5

[0419] -50- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT

[0420] leads to the removing the inhibitory signal that CD5 can transmit in activated T-Cells. Thus, the down regulation of surface expression allows for the T-Cells to be more active, which in turn leads to an increase in IFNy secretion.

[0421] Example 6: Cross-linked antibodies induce surface CD5 downregulation. To better mimic a bispecific antibody, anti-CD5 antibodies and other antibodies were cross-linked with BS3. Briefly, cells were treated overnight with either Transact or 0.25 pg / mL BS3-conjugated (BS3, Thermo Scientific, #A39266), soluble antibodies, as indicated [TransAct, (Miltenyi, #130111-160); mlgGl isotype (Invitrogen, #02-6100), CD5 AB2, and CD7 AB1], Following treatment, cells were stained with UCHT2-APC (1:100; anti-CD5, BioLegend, #300612) or BLla-PacificBlue (1:50; anti-CD5, Beckman Coulter, #A82790), and CD5 surface expression was analyzed by flow cytometry. As illustrated in FIG. 9A and 9B, the cross-linked anti-CD5 antibody (BS3-CD5 AB2) or the cross-linked anti-CD5 / anti-CD7 antibody combination (BS3-CD5 AB2-CD7 AB1) lead to a decrease in CD5 surface expression. Thus, without being bound to any particular theory, the internalization of CD5, which can be mediated by a bispecific-like antibody leads to the removing the inhibitory signal that CD5 can transmit in activated T-Cells. Thus, the down regulation of surface expression allows for the T-Cells to be more active, which in turn leads to an increase in IFNy secretion.

[0422] Thus, the embodiments and examples provided for herein demonstrate that a tethered an anti-CD5 molecule, such as in bispecific format, unexpectedly leads to the enhancement and activation of CAR-T cells. This effect was surprising and unexpected. Additionally, the embodiments and examples provided for herein demonstrate that a multi-specific polypeptide that binds to the extracellular domain of CD5 and at least one other immune cell surface protein can be used to enhance cytotoxic killing of tumor cells and can be used to treat various cancers or other diseases as provided for herein. The results also demonstrate the ability of such polypeptides to enhance the effectiveness of CAR-T therapy. These effects were surprising and could not have been predicted.

[0423] Example 7: Expression of anti-CD5 scFv on surface of T cell leads to down regulation of CD5 expression

[0424] Given the above results with respect to tethered anti-CD5 antibodies, it was investigated whether expression of an anti-CD5 molecule on the surface of a T cell would lead to a similar down regulation CD5 expression on the surface of the T cell. Cells were -51- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT

[0425] transduced with a CAR19 construct, a first anti-CD5 scFv construct (CD5 AB1), a second anti-scFv construct (CD5 AB2), or combinations thereof and the surface expression of CD5 on T cells was assessed. As shown in FIG. 10, un-transduced cells (UTD) and cells transduced solely with the CD 19 CAR (CAR19) served as controls and exhibited no change in CD5 surface expression. Transduction of T cells with the first surface anti-CD5 scFv construct (CD5 AB1) did not alter the expression of CD5 on the surface of the T cells.

[0426] However, transduction of T cells with the second anti-CD5 scFv construct (CD5 AB2) resulted in a marked reduction of surface CD5 expression in the T cells. The co-expression of both anti-CD5 scFv constructs also resulted in a marked reduction in CD5 expression, likely driven by the CD5 AB2 construct. To determine if expression of the CAR19 construct altered the effect of the anti-CD5 scFvs, T cells were transduced with both a CAR 19 and either the CD5 AB2 anti-CD5 scFv or the CD5 AB1 anti-CD5 scFv. In both cases, expression of the CAR19 did not significantly alter the CD5 expression pattern observed with the anti-CD5 scFv constructs alone.

[0427] As shown in FIG. 11, the difference in effect of the CD5 AB2 and CD5 AB1 anti-CD5 scFv constructs was not due to a difference in transduction efficiency. Both constructs resulted in similar anti-CD5 scFv expression levels, whether singly transduced, transduced in combination, or transduced in combination with the CAR19 construct. Additionally, as shown in FIG. 12, the CD5 AB2 anti-CD5 scFv does not negatively impact the expression of the CAR19, but rather specifically down regulates CD5 expression.

[0428] The results of this example provide the surprising and unexpected result that certain anti-CD5 scFv constructs expressed on the surface of a T cell will result in the down regulation of CD5 in that T cell.

[0429] Example 8: CD5 down regulation the result of specific epitope targeting of anti-CD5 scFv

[0430] To elucidate why certain anti-CD5 scFv constructs result in down regulation of surface CD5 on T cells but not others, a panel of anti-CD5 scFvs targeting specific epitopes of CD5 are tested. Surprisingly, unique anti-CD5 scFvs targeting the same epitope on CD5 as CD5 AB2 result in a similar down regulation of CD5, indicating that targeting specific epitopes of CD5 are key to its down regulation.

[0431] Example 9: Soluble anti-CD5 antibodies drive surface CD5 internalization in TransAct-activated CAR T cells.

[0432] -52- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT

[0433] To further investigate the observations detailed above, additional anti-CD5 antibodies were interrogated. The table below provides the antibodies and control IgGs utilized in Examples 9-13 below.

[0434] Antibody Species Origin Isotype Fc Function

[0435] mlgGl Mouse IgGl Wild type

[0436] mlgG2a Mouse lgG2a Wild type

[0437] CD7AB1 Mouse IgGl Wild type

[0438] CD5 AB1 Mouse IgGl Wild type

[0439] CD5 AB 2 Mouse IgGl Wild type

[0440] CD5 AB 3 Mouse IgGl Wild type

[0441] CD5 AB4 Mouse lgG2a Wild type

[0442] hlgGl Human IgGl Wild type

[0443] CD5 AB 5 Humanized IgGl Effectorless

[0444] CD5 AB 6 Humanized IgGl Effectorless

[0445] CD5 AB7 Humanized IgGl Effectorless

[0446] CD5 AB 8 Humanized IgGl Effectorless

[0447] CD5 AB 9 Humanized IgGl Wild type

[0448] MHC 1 antibody Mouse IgGl Wild type

[0449] OKT3 Mouse lgG2a Wild type

[0450]

[0451] First, select antibodies were assessed for their efficiency in driving surface CD5 internalization in TransAct-activated CART cells. Briefly, TransAct-activated CAR T cells expressing wild-type CD5 were plated in flat-bottom 96-well plates at 100k cells per well in 100 pL RPMI. Anti-CD5 antibodies (500 ng per well) were added to a final volume of 200 pL, followed by a 2-hour incubation at 37°C in a CO₂ incubator. Cells were then washed and stained with anti-CD5 BL1a–PE and UCHT2–APC antibodies before analysis by flow cytometry. Data were processed using FlowJo software and are displayed as histograms with half-offset overlays. The data are shown in FIGs. 13A and 13B.

[0452] As illustrated in FIG. 13A and 13B, anti-CD5 antibody CD5 AB2 drove surface CD5 internalization as measured both by BL1 a and UCHT2 staining. This is in agreement with previous results shown in FIG. 8 and FIG. 10. In contrast, anti-CD5 antibodies CD5 AB3 and CD5 AB4 do not effectively drive internalization of surface CD5. As shown in FIG.

[0453] -53- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT

[0454] 13A, surface expression of CD5 is identical in untreated and CD5 AB3 treated cells. The reduction in signal shown in FIG. 13B is likely due to the treatment (CD5 AB3) and detection antibodies (UCHT2) binding to the same epitope, i.e., the treatment antibody is binding surface CD5 and blocking the detection antibody from binding. Similar results are obtained for anti-CD5 antibody CD5 AB4. As shown in FIG. 13B, CD5 AB4 treatment does not reduce surface CD5 expression as assessed by UCHT2 binding. The large decrease in expression shown in FIG. 13A is likely due to the antibodies BLla and CD5 AB4 competing for the same epitope on CD5.

[0455] The results of this example show that only select anti-CD5 antibodies are capable of driving surface CD5 internalization in TransAct-activated CART cells.

[0456] Example 10: Tethered anti-CD3 antibody OKT3 enhance IFN gamma production in PBMCs as compared to soluble antibody.

[0457] It was next assessed what effect tethered anti-CD3 antibodies would have on primary PBMCs. Briefly, peripheral blood mononuclear cells (PBMCs) from two healthy donors were stimulated with either plate-bound or soluble OKT3 antibody (anti-CD3) for 72 hours. For coated OKT3, flat -bottom 96-well plates were pre-coated with serial dilutions of OKT3 antibody (mouse IgG2a) in 100 pL per well and incubated overnight at 4 °C. Wells were washed with PBS and blocked with 1% BSA in PBS (200 pL per well) overnight at 4 °C. Blocking buffer was removed immediately prior to cell addition. PBMCs were added at cells per well in 100 pL RPMI, followed by 100 pL PBS to reach a final volume of 200 pL. For soluble OKT3, antibody was added directly to wells at matched concentrations in 100 pL RPMI, followed by 100 pL of PBMCs at cells per well. After 72 hours of incubation at 37 °C in a CO₂ incubator, supernatants were collected and IFNγ levels were quantified using the IFNγ ELISA kit (Thermo Fisher Scientific). The results are shown in FIGs. 14A and 14B.

[0458] As shown in FIGs. 14A and 14B, soluble OKT3 antibody provided a low level TCR / CD3 complex activation as measured by IFNy production that remained relatively constant irrespective of antibody concentration. In contrast, tethered OKT3 antibody resulted in increased IFNγ production in a dose dependent manner. Additionally, for each donor, at the higher concentrations of antibody tested, tethered antibody showed a drastic increase in IFNγ production as compared soluble antibody.

[0459] Accordingly, the results of this example illustrate that OKT3 antibody was able to activate primary PBMCs, and that tethered OKT3 antibody produced greater activation at -54- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT

[0460] higher concentrations. The results with antibody tethering, mimicking a bispecific antibody, suggest that an anti-CD3 binding bispecific antibody may result in greater TCR / CD3 complex activation in PBMCs.

[0461] Example 11: Soluble anti-CD5 antibodies synergistically enhance IFN gamma production in PBMCs when administered with anti-CD3 antibodies.

[0462] Having shown that different CD5 antibodies targeting different epitopes on CD5 have differential effects on the internalization of surface CD5 and on cell activation, it was next assessed whether co-administration of the anti-CD3 antibody OKT3 with various anti-CD3 antibodies would enhance activation of PBMCs. Briefly, peripheral blood mononuclear cells (PBMCs) from one healthy donor were stimulated for 72 hours under four experimental conditions: A. No stimulation (No ab control); B. Soluble OKT3 alone (50ng / mL); C.

[0463] Soluble anti-CD5 antibody alone (1 pg / mL); D. Combined stimulation with soluble OKT3 (50 ng / mL) plus either anti-CD5 antibody or isotype control (1 pg / mL). Antibodies were added directly to wells in 100 pL RPMI, followed by 100 pL of PBMCs at 100k cells per well. Cultures were incubated at 37 °C in a CO₂ incubator for 72 hours. Supernatants were harvested and IFNγ levels were quantified using the IFNγ ELISA kit (Thermo Fisher Scientific). Results are shown in FIG. 15.

[0464] As shown in FIG. 15, treatment of PBMCs with soluble OKT3 antibody alone resulted in a low level of TCR / CD3 activation as expected, as shown by IFNγ measurements. Also as expected, none of the anti-CD5 antibodies tested resulted in TCR / CD3 complex activation on their own. However, for each anti-CD5 antibody tested, the combination of the anti-CD3 and anti-CD5 antibodies resulted in a synergistic enhancement of TCR / CD3 complex activation. The enhanced activation was greater than what was observed for the isotype control condition, indicating that the results are not an artifact of the isotype.

[0465] Interestingly, each antibody tested binds to a unique epitope on CD5, and only CD5 AB2 results in internalization of surface CD5 (See FIGs 8, 10, and 13A and B). Thus, internalization and degradation of surface CD5 is not a requirement for the enhanced activation of PBMCs. Although, CD5 AB2 did show the most robust synergistic effect of antibodies tested.

[0466] These results suggest that binding of surface CD5 with anti-CD5 antibodies can enhance anti-CD3 antibody mediated PBMC activation, resulting in a gain of function for the anti-CD3 antibody. The combination of anti-CD5 antibodies with anti-CD3 antibodies could

[0467] -55- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT

[0468] thus potentially be useful as a therapeutic due to the synergistic activation of the TCR / CD3 complex.

[0469] Example 12: Expanded panel of soluble anti-CD5 antibodies shows additional anti-CD5 antibodies capable of driving surface CD5 internalization in TransAct-activated CAR T cells.

[0470] An expanded panel of anti-CD5 antibodies was tested for their ability to drive surface CD5 internalization. Briefly, TransAct-activated CAR T cells expressing wild-type CD5 were plated in flat-bottom 96-well plates at 100k cells per well in 100 pL RPMI. Anti-CD5 antibodies (500 ng per well) were added to a final volume of 200 pL, followed by a 2-hour incubation at 37 °C in a CO₂ incubator. Cells were then washed and stained with anti-CD5 BL1a–PE and UCHT2–APC antibodies before analysis by flow cytometry. Data were processed using FlowJo software and are displayed as histograms with half-offset overlays. The results are illustrated in FIG. 16A and 16B.

[0471] As shown in FIG. 16A and 16B, additional antibodies were identified that were capable of driving surface CD5 internalization to varying degrees. As discussed in Example 9, antibodies that show decreased surface CD5 with one detection antibody and not the other detection antibody likely do not drive internalization of surface CD5, but rather likely compete for binding at the same epitope as one of the detection antibodies. Anti-CD5 antibody CD5 AB 2 showed decreased surface CD5 expression under both detection antibodies, agreeing with the previous results of Examples 5, 7, and 9. Antibodies CD5 AB9 and CD5 AD5 also exhibited decreased surface CD5 expression under both detection antibodies, indicating that these two antibodies likely also decrease surface CD5 expression.

[0472] These results illustrate that additional anti-CD5 antibodies are capable of inducing surface CD5 internalization in a similar manner to antibody CD5 AB2.

[0473] Example 13: Expanded panel of soluble anti-CD5 antibodies synergistically enhance IFN gamma production in PBMCs when administered with anti-CD3 antibodies.

[0474] The expanded panel of anti-CD5 antibodies was next tested for their ability to act in a synergistic manner with anti-CD3 antibodies in a similar manner as described in Example 11. Briefly, peripheral blood mononuclear cells (PBMCs) from two healthy donors were plated at cells per well in 96-well flat-bottom plates containing soluble OKT3 antibody at 125 ng / well. After a 30-minute incubation at 37 °C in a CO₂ incubator, the following conditions were applied: A. PBMCs alone (no antibody); B. Soluble OKT3 alone; C. Soluble OKT3 plus CD5

[0475] -56- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT

[0476] antibody; D. Soluble OKT3 plus CD5 antibody followed by goat anti-human Fc polyclonal antibody at a 1:2 molar ratio (Fc antibody in 2-fold excess): E. Soluble OKT3 plus CD5 antibody with goat anti -human Fc antibody pre-saturated by 4-fold molar excess of human IgGl (hlgGl) at room temperature prior to addition. After treatment, cells were incubated for 72 hours at 37 °C. Supernatants were collected and IFNy levels were quantified using the IFNy ELISA kit (Thermo Fisher Scientific). The results are shown in FIG. 17A and 17B.

[0477] As shown in FIG. 17A, OKT3 antibody alone resulted in a low level of TCR / CD3 activation as exxpected, as shown by IFNγ measurements. For each of the new anti-CD5 antibodies tested, combination of the anti-CD5 antibody with the OKT3 antibody produced a greater level of TCR / CD3 activation than with the OKT3 antibody alone, in agreement with the results of Example 11. Addition of the goat anti-human Fc antibody after treatment with the anti-CD3 / anti-CD5 antibody combination resulted in an even greater TCR / CD3 activation for all antibody combinations tested. Blocking the cells with a 4 fold molar excess of goat anti-human Fc prior to addition of the anti-CD3 / anti-CD5 antibody combination blunted the effect observed when the goat anti-human Fc antibody was added after anti-CD3 / anti-CD5 antibody treatment for all combinations.

[0478] FIG. 17B shows similar results for a separate PBMC donor. In each case, the combination of the anti-CD5 antibody with the OKT3 antibody produced a greater level of TCR / CD3 activation than with the OKT3 antibody alone. In three of five conditions tested, addition of the goat anti-human Fc antibody after treatment with the anti-CD3 / anti-CD5 antibody combination resulted in an even greater TCR / CD3 activation, in agreement with the results from the first donor. Blocking with the goat anti-human FC antibody blunted the synergistic effect of the anti-CD3 / anti-CD5 antibody combination in four of the five conditions tested.

[0479] The results of this example illustrate that a multivalent CD5 binder can result in even greater gain of function for anti-CD3 antibody mediated activation of the TCR / CD3 complex. In the present example, addition of the goat anti-human Fc after antibody treatment acts to create a multivalent anti-CD5 antibody complex due to the Fc binding the anti-CD5 antibodies. This mimics an anti-CD5 bispecific antibody. As the results show, the multivalent anti-CD5 antibody complex results in even greater activation of the TCR / CD3 complex, as measured by IFNγ production. Thus, multivalent CD5 binders, such as a multivalent anti-CD5 bispecific antibody, could be used as a therapeutic by enhancing TCR activation.

[0480] -57- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT

[0481] The disclosures of each and every patent, patent application, accession number, and publication cited herein are hereby incorporated herein by reference in their entirety. While various embodiments have been disclosed with reference to specific aspects, it is apparent that other aspects and variations of these embodiments may be devised by others skilled in the art without departing from the true spirit and scope of the embodiments. The appended claims are intended to be construed to include all such aspects and equivalent variations.

[0482] -58- IPTS / 200305043.1

Claims

DOCKET NO. VTB-013WO PATENTWhat is claimed:

1. A polypeptide comprising a first binding domain (effector moiety) and a second binding domain (effector moiety), wherein:the first binding domain binds to an extracellular domain of CD5; and the second binding domain binds to an immune cell surface protein.

2. The polypeptide of claim 1, wherein the second binding domain binds to an extracellular domain of CD5.

3. The polypeptide of claim 2, wherein the first binding domain and the second binding domain bind to the same or different CD5 epitopes.

4. The polypeptide of claim 2, wherein the first binding domain and the second binding domain bind to the same CD5 epitope.

5. The polypeptide of any one of claims 1-4, wherein the first binding domain binds to the DI domain, the DII domain, or the Dill domain of the extracellular domain of CD5 and the second binding domain binds to the DI domain, the DII domain, or the Dill domain of the extracellular domain of CD5, wherein the first binding domain and second binding domain bind to the same or different domains.

6. The polypeptide of claim 5, wherein the first binding domain and the second binding domain do not bind to the same domain of DI, DII, or Dill of CD5.

7. The polypeptide of any one of claims 1-6, wherein the first binding domain binds to DI, DII, Dill, the linker between DI and DII, the linker between DII and Dill, or the linker between Dill and the transmembrane domain of the CD5.

8. The polypeptide of any one of claims 1-7, wherein the second binding domain binds to DI, DII, Dill, the linker between DI and DII, the linker between DII and Dill, or the linker between Dill and the transmembrane domain of the CD5.

9. The polypeptide of claim 1, wherein:-59- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENTthe first binding domain binds to DI, DII, Dill, the linker between DI and DII, the linker between DII and Dill, or the linker between Dill and the transmembrane domain of the CD5; andthe second binding domain binds to DI, DII, Dill, the linker between DI and DII, the linker between DII and Dill, or the linker between Dill and the transmembrane domain of the CD5,provided that the first and second binding domains do not bind to the same domain, linker, or the same epitope.

10. The polypeptide of any one of claims 1-9, wherein the first binding domain binds to DI domain of CD5 and the second binding domain binds to Dill domain of CD5.

11. The polypeptide of any one of claims 1-10, wherein the first binding domain when bound to CD5 leads to its internalization or degradation of CD5.

12. The polypeptide of claim 1, wherein the second binding domain binds to a Pan T-Cell surface protein.

13. The polypeptide of claim 12, wherein the Pan T-Cell surface protein is PD-1, CD7, CD2, CD3, PD-1, CD-28, TCR, MHC, CD4, CD8, TIGIT, CD25, CCR5, CXCR4, CDla, CD28, CTLA-4, ICOS, 4-1BB (CD137), LFA-1, VLA-4, CD44, PSGL-1, CD6, CD40L, CD45RO, CD45RA, CD62L, CXCR3, CCR7, CD27, Fas (CD95), CD38, LAG-3, TIM-3, CD16, CD56, or CD107.

14. The polypeptide of claim 13, wherein the Pan T-Cell surface protein is CD7 or CD3.

15. The polypeptide of any one of claims 1-11, wherein the second binding domain binds to a recombinant engineered T cell specific target cell antigen.

16. The polypeptide of claim 15, wherein the recombinant engineered T cell specific target cell antigen is peptide linker (e.g., glycine / serine, and the like) or directed to a nonnative antibody sequence present on the surface of the T cell, such as, but not limited to the antigen binding domain of a chimeric antigen receptor present on the surface of an engineered T cell.-60- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT17. The polypeptide of any one of claims 1-16, wherein the first binding domain is an antibody or ligand that binds to CD5 and the second binding domain is an antibody or ligand that binds to the immune cell surface protein or the recombinant engineered T cell specific target cell antigen.

18. The polypeptide of claim 17, wherein the first binding domain is a scFv antibody or a Fab antibody and the second binding domain is a scFv antibody or a Fab antibody.

19. The polypeptide of claim 17, wherein the first binding domain is an scFv antibody and the second binding domain is an scFv antibody.

20. The polypeptide of claim 17, wherein the first binding domain is an scFv antibody and the second binding domain is a Fab antibody.

21. The polypeptide of claim 17, wherein the first binding domain is a Fab antibody and the second binding domain is a Fab antibody.

22. The polypeptide of claim 17, wherein the first binding domain is a Fab antibody and the second binding domain is an scFv antibody.

23. The polypeptide of any one of claims 1-22, wherein the polypeptide comprises a third binding domain (effector moiety) that binds to a cell specific antigen, such as a target cell or tumor antigen, or an immune cell surface antigen that is a different protein from what the first binding domain and the second binding domain binds to.

24. The polypeptide of claim 23, wherein the target cell antigen is CD 19, CD22, TAG-72, MUC16, PSMA, EGFR, a-integrin, BCMA, HER2, Mesothelin, CLDN6, Nectin-4, CEA, and the like.

25. The polypeptide of any one of claims 1-22, wherein the polypeptide comprises a third binding domain (effector moiety) that binds to a recombinant engineered T cell specific target-61- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENTcell antigen, provided that the second binding domain binds to an immune cell surface protein that is not the recombinant engineered T cell specific target cell antigen.

26. The polypeptide of claim 25, wherein the recombinant engineered T cell specific target cell antigen is peptide linker (e.g., glycine / serine, and the like) or directed to a nonnative antibody sequence present on the surface of the T cell, such as, but not limited to the antigen binding domain of a chimeric antigen receptor present on the surface of an engineered T cell.

27. A pharmaceutical composition comprising the polypeptide of any one of claims 1-26 and a pharmaceutically acceptable excipient.

28. A nucleic acid molecule or molecules encoding the polypeptide of any one of claims 1-26.

29. A host cell comprising the nucleic acid molecule or molecules of claim 28.

30. A method of producing the polypeptide of any one of claims 1-26, the method comprising culturing a host cell comprising one or more nucleic acid molecules encoding the polypeptide under conditions to express and produce the polypeptide.

31. The method of claim 30, wherein the method further comprises isolating the expressed polypeptide.

32. A method of treating cancer or an auto-immune disease in a subject, the method comprising administering to the subject a pharmaceutical composition comprising the polypeptide of any one of claims 1 -26.

33. The method of claim 32, wherein the subject has previously been treated with CAR-T therapy.

34. The method of claim 33, wherein the CAR-T therapy is ex-vivo CAR-T therapy or in-vivo CAR-T therapy.-62- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT35. The method of claim 32, wherein the subject has not been previously treated with CAR-T therapy.

36. The method of claim 32, wherein the method comprises co-administering the polypeptide and an additional cancer therapeutic.

37. The method of claim 36, wherein the additional cell therapeutic is an engineered cell therapy or gene therapy to treat the cancer.

38. The method of claim 37, wherein the engineered cell therapy is CAR-T cell therapy.

39. The method of claim 38, wherein the CAR-T cell therapy is ex-vivo CAR-T cell therapy or in-vivo CAR-T cell therapy.

40. The method of any one of claims 32-39, wherein the cancer is not a CD5 positive cancer.

41. The method of claim 40, wherein the cancer is not T-cell lymphoma.

42. A method of reducing CD5 surface expression on an immune cell, the method comprising contacting the immune cell expressing CD5 on its surface with a pharmaceutical composition comprising the polypeptide of any one of claims 1-26.

43. The method of claim 42, wherein the immune cell is a CAR-T cell.

44. The method of claim 42 or 43, wherein the method comprises administering to a subject comprising the immune cell the pharmaceutical composition.

45. A method of activating a T-cell, the method comprising contacting a T cell expressing CD5 on its surface with a pharmaceutical composition comprising the polypeptide of any one of claims 1-26.

46. The method of claim 45, wherein the immune cell is a CAR-T cell.-63- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT47. The method of claim 45 or 46, wherein the method comprises administering to a subject comprising the T cell the pharmaceutical composition.

48. A T-cell comprising a chimeric antigen receptor and a polypeptide expressed on the surface of the T-cell, wherein the polypeptide expressed on the surface of the T-cell comprises an antigen binding domain that binds to a surface protein of the T-cell.

49. The T-cell of claim 48, wherein the antigen binding domain binds to CD5.

50. The T-cell of claim 49, wherein the antigen binding domain that binds to CD5 comprises an scFv.

51. The T-cell of claim 49 or 50, wherein the antigen binding domain that binds to CD5 binds to the same epitope as an anti-CD5 antibody, or antigen binding fragment thereof, comprising a variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 7 and a variable light chain domain comprising the amino acid sequence of SEQ ID NO: 8.

52. The T-cell of any one of claims 49-51, wherein the antigen binding domain that binds to CD5 binds to the same epitope as an anti-CD5 scFv comprising the amino acid sequence of SEQ ID NO: 9.

53. The T-cell of any one of claims 49-52, wherein the antigen binding domain that binds to CD5 binds to the extracellular domain of CD5.

54. The T-cell of claim 53, wherein the binding to the extracellular domain of CD5 results in internalization of the CD5.

55. The T-cell of claim 53, wherein the binding to the extracellular domain of CD5 results in degradation of the extracellular domain of CD5, the cytoplasmic domain of CD5, or both the extracellular domain of CD5 and the cytoplasmic domain of CD5.

56. The T-cell of claim 53, wherein the binding to the extracellular domain of CD5 leads to the cleavage of the extracellular domain of CD5.-64- IPTS / 200305043.1DOCKET NO. VTB-013WO PATENT57. The T-cell of any one of claims 48-56 wherein the T-cell is a CD5 positive T-cell.

58. The T-cell of any one of claims 48-57, wherein the chimeric antigen receptor comprises an antigen binding domain that binds to CD5.

59. The T-cell of any one of claims 48-57, wherein the chimeric antigen receptor comprises an antigen binding domain that does not bind to CD5.-65- IPTS / 200305043.1

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