Novel ceacam5 binding agents and methods of treatment

Description

NOVEL CEACAM5 BINDING AGENTS AND METHODS OF TREATMENTCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to United States Provisional Patent Application No. 63 / 616,293, filed on December 29, 2023, the entire content of which is incorporated herein by reference.FIELD

[0002] The present disclosure relates to novel CEACAM5 binding agents and methods of their use in the treatment of cancer.BACKGROUND

[0003] CEACAM5 (carcinoembryonic antigen-related cell adhesion molecule 5) is a glycophosphatidylinositol-anchored membrane protein and established tumor antigen whose expression has primarily been associated with adenocarcinomas of the colon, rectum, and pancreas. To date, no CEACAM5 binding agents have been utilized for any approved therapeutic use.

[0004] It is therefore of great interest to develop improved CEACAM5 directed compositions in order to facilitate treatment of cancers associated with high level expression of CEACAM5.SUMMARY OF THE INVENTION

[0005] The present disclosure is based, at least in part, on the development of reduced affinity CEACAM5 binding agents (such as antibodies) with variant heavy chain variable (VH) region CDR3 domains providing advantages over existing anti-CEACAM5 VH CDR3 domains. For example, the variant CDR3 domains disclosed herein exhibit a reduced binding affinity to CEACAM5 as compared to their corresponding binding domains in the parental antibodies from which they were derived. The compositions and cells comprising these CEACAM5 binding agents are designed to promote reduced T cell exhaustion, improved in vivo persistence, improved tumor cell killing, and reduced toxicity in normal tissues following their administration when applied to the therapeutic binding agents disclosed herein. In addition, when used in combination with other binding domains in a multi- specific format, the CEACAM5 binding agents can advantageously bridge tumor cells and immune cells toprovide enhanced anti-tumor responses and / or avidity-driven crosslinking of co-expressed tumor-associated antigens (TAAs).

[0006] In one embodiment, the CEACAM5 binding agent comprises a CEACAM5 binding domain comprising a heavy chain variable (VH) region comprising complementary determining 1 (HC CDR1), complementary determining 2 (HC CDR2), and complementary determining 3 (HC CDR3) regions; and a light chain variable (VL) region comprising complementary determining 1 (LC CDR1), complementary determining 2 (LC CDR2), and complementary determining 3 (LC CDR3) regions, where the VH region comprises HC CDR1 and HC CDR2 regions comprising amino acid sequences set forth SEQ ID NOs: 1 and 2, respectively, and an HC CDR3 region comprising an amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 4; and where the VL region comprises LC CDR1, LC CDR2, and LC CDR3 regions comprising amino acid sequences set forth in SEQ ID NOs: 5-7, respectively.

[0007] In another embodiment, the VH region comprises HC CDR1 and HC CDR2 regions comprising amino acid sequences set forth in SEQ ID NOs: 34 and 35, respectively, an HC CDR3 region comprising an amino acid sequence set forth in SEQ ID NO: 36 or 37; and the VL region comprises LC CDR1, LC CDR2, and LC CDR3 regions comprising amino acid sequences set forth in SEQ ID NOs: 38-40, respectively.

[0008] In another embodiment, the VH and VL region amino acid sequences are selected from the group consisting of: (a) SEQ ID NOs: 9 and 11, respectively; or (b) SEQ ID NOs: 10 and 11, respectively; (c) SEQ ID NOs: 42 and 43, respectively; and (d) SEQ ID NOs: 43 and 44, respectively.

[0009] In another embodiment, the CEACAM5 binding agent is a monospecific or multispecific single chain polypeptide. In one embodiment, the monospecific single chain polypeptide is an anti-CEACAM5 single chain variable fragment (used herein interchangeably with the term CEACAM5 scFv). In another embodiment, the monospecific single chain polypeptide is a CEACAM5 scFv fused to a monomeric immunoglobulin IgG constant region domain capable of pH-dependent binding to FcRn in order to provide extended in vivo halflife. Exemplary constant region domains include CH2, CH3, CH1-CH2, CH1-CH3, and CH1- CH2-CH3 polypeptide chains.

[0010] In another embodiment, the CEACAM5 binding agent of the present disclosure comprises a flexible linker between one or more variable regions or a variable region and one or more Fc region components. In another embodiment, the linker is positioned between the VH and VL regions in e.g., a CEACAM5 scFv. In one embodiment, the linkercomprises the amino acid sequence (GxS)n in which x is an integer between 1-6, inclusive, and n is an integer between 1-10, inclusive. In another embodiment, the linker comprises the amino acid sequence (GGGS)n (SEQ ID NO: 67) in which n is an integer between 1-10, inclusive. In another embodiment, the linker comprises the amino acid sequence multimer GGGGSGGGGSGGGGS (SEQ ID NO: 68). In one embodiment, the CEACAM5 binding agent comprises an CEACAM5 scFv comprising an amino acid sequence set forth in any one of SEQ ID NOs: 17, 18, 50, or 51.

[0011] In another embodiment, the CEACAM5 binding agent is an IgG. In another embodiment, the IgG is selected from the group consisting of an IgGl, an IgG2, an IgG3, an IgG4 or a variant thereof.

[0012] In another embodiment, the CEACAM5 binding agent is a bispecific CEACAM5 binding agent comprising the CEACAM5 binding domain and a second antigen binding domain. In another embodiment, the CEACAM5 binding agent is a bispecific single chain polypeptide, such as an scFv2, an scFv2-Fc, a tandem scFv (ta-scFv), a single chain Fab (scFab) fragment, a single chain diabody (scDb), a disulfide-bond stabilized scFv (ds-scFv), an scFv-Fc, or a tandem diabody (TandAb). In another embodiment, the CEACAM5 binding agent is a bispecific double chain polypeptide, such as a dual-affinity retargeting (DART). In another embodiment, the bispecific CEACAM5 binding agent is a bispecific scFv (bi-scFv), an scFv-Fab, an scFv-scFab, a cross-over F(ab), a F(ab’)2, a diabody, a tandem diabody, or a minibody.

[0013] In another embodiment, the second antigen binding domain is an scFv. In another embodiment, the second antigen binding domain binds a target antigen selected from the group consisting of ICAM-1, c-MET, EpCAM, and SSTR2. In another embodiment, the second antigen binding domain comprises an scFv having an amino acid sequence set forth herein.

[0014] In another embodiment, the bispecific CEACAM5 binding agent is a bispecific immune cell engager. In another embodiment, the bispecific CEACAM5 binding agent is a bispecific T cell engager (BiTE). In one embodiment, the BiTE comprises a CD3 binding domain. In another embodiment, the bispecific CEACAM5 binding agent is a bispecific natural killer (NK) cell engager (NKCE). In one embodiment, the NKCE comprises a Fey receptor Illa (or CD 16a) binding domain and / or an NKp46 binding domain. In another embodiment, the bispecific CEACAM5 binding agent is a bispecific macrophage engager (BiME). In one embodiment, the BiME comprises a signal regulatory protein-a (SIRPa)-, aCD47-, a CD89-, and / or a Mer proto-oncogene tyrosine kinase (MerTK) receptor binding domain.

[0015] In another embodiment, the CEACAM5 binding agent is a multi- specific binding agent comprising the CEACAM5 binding domain, such as a trispecific binding agent comprising two or more additional antigen binding domains. In one embodiment, the CEACAM5 binding agent is a trispecific antibody comprising a CEACAM5 binding domain, a second antigen binding domain, and a third antigen domain.

[0016] In some embodiments, the EC50 of the CEACAM5 binding domain is greater than 5 pM, greater than 10 p M, greater than 15 pM, greater than 20 pM, greater than 25 pM, greater than 30 pM, greater than 35 pM, greater than 40 pM, greater than 50 pM, greater than 5 pM but less than 50 pM, or any range thereof. In other embodiments, the fold change in EC50 relative to a corresponding wild-type CEACAM5 binding domain is greater than 500-fold, greater than 800-fold, greater than 1,000-fold, greater than 2,000-fold, greater than 5,000-fold, greater than 10,000-fold, greater than 20,000-fold, greater than 30,000-fold, greater than 800-fold but less than 30,000-fold, or any range thereof.

[0017] In another embodiment, the bispecific CEACAM5 binding agent further comprises an immunoglobulin (Ig) constant region domain, an Fc domain, or an albumin binding region. In another embodiment, the immunoglobulin constant region comprises an IgGl CHI domain, an IgGl CH2 domain, an IgGl CH3 region, or a combination thereof. In another embodiment, the Fc domain comprises a monomeric single chain Fc fragment (sc(Fc)i or scCH2-CH3). In another embodiment, the Fc domain comprises a dimeric single chain Fc fragment (sc(Fc)2).

[0018] In another embodiment, the Fc domain comprises a mutant constant region domain or mutant Fc region domain genetically engineered to provide enhanced antibody dependent cellular cytotoxicity. In another embodiment, the Fc domain comprises a mutant constant region domain or mutant Fc region domain genetically engineered to possess reduced effector functions or a lack of effector functions (effector less).

[0019] In another embodiment, the CEACAM5 binding agent consists of a single polypeptide chain. In another embodiment, the CEACAM5 binding agent consists of two polypeptide chains. In another embodiment, a first polypeptide chain and a second polypeptidechain are linked by one or more disulfide bonds. In another embodiment, the first polypeptide chain and the second polypeptide chain are linked by VH-VL pairing.

[0020] In another embodiment, each of the first and second polypeptide chains comprises an Fc fragment having complementary mutations between one another that enhance heterodimerization over homodimerization so as to force the association of the first polypeptide chain comprising the CEACAM5 binding domain to a second polypeptide chain comprising the second binding domain. In another embodiment, the complementary mutations are knobhole mutations. In a further embodiment, the knob mutations are selected from S354C, T366W and / or K409A, and the hole mutation are selected from S354C, Y349C, T366S, L368A, F405K, and / or Y407V.

[0021] In another aspect, the present disclosure provides an antibody-drug conjugate (ADC) comprising a cytotoxic drug, a linker, and a CEACAM5 binding agent described herein. In another embodiment, the cytotoxic drug is a maytansinoid, a camptothecin, a dolastatin, an aurastatin, a pyrrolobenzodiazepine (PBD), a calicheamicin, a duocarmycin, a tubulolysin, an analogue thereof, or a combination thereof. In another embodiment, the cytotoxic drug is deruxtecan, exatecan, SN-38, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAE), Auristatin F-hydroxypropylamide, AurOlOl, emtansine (DM1), Maytansinoid DM4, duocarmycin, duostatin-5, or a combination thereof.

[0022] In another embodiment, the linker is a cleavable linker, such as maleimidocaproyl-L-valine-L-citrulline-p-aminobenzoyl carbamate (mc-VC-PABC) or malcimido-dPEG8-L-valinc-L-alalinc- -aminobcnzoyl carbamate (M-dPEG8-VA-PABC). In another embodiment, the linker is a non-cleavable linker, such as maleimidocaproyl (MC), succinimidyl-4-(N-maleimidomethyl)cyclohexane-l -carboxylate (SMCC), or a thioether- containing linker.

[0023] In another embodiment, the ADC has a drug-to-antibody (DAR) ratio between 2-10.

[0024] In another aspect, the present disclosure provides a nucleic acid or set of nucleic acids encoding a CEACAM5 binding domain comprising a VH region and a VL region, where the VH and VL regions are encoded by nucleotide sequences selected from the group consisting of: (a) SEQ ID NOs: 13 and 15, respectively; (b) SEQ ID NOs: 14 and 15,respectively; (c) SEQ ID NOs: 46 and 48, respectively; and (f) SEQ ID NOs: 47 and 48, respectively.

[0025] In another embodiment, the nucleic acid or set of nucleic acids encode a CEACAM5 scFv and a second scFv binding a different target antigen, where the CEACAM5 scFv and / or second scFv comprises a (GxS)nlinker between the VH and VL regions. In one embodiment, the nucleic acid comprises a polynucleotide sequence encoding a CEACAM5- scFv comprising an amino acid sequence set forth in in any one of SEQ ID NOs: 17, 18, 50, or 51. In another embodiment, the nucleic acid encoding the CEACAM5-scFv comprises a polynucleotide sequence set forth in any one of SEQ ID NOs: 20, 21, 53, or 54.

[0026] In another embodiment, the nucleic acid or set of nucleic acids encoding the CEACAM5 binding agent include one or more polynucleotide sequences encoding a selfcleaving 2A peptide from porcine teschovirus- 1 (P2A), equine rhinitis A virus (E2A), thosea asigna virus (T2A), or foot-and-mouth disease virus (F2A), or a combination thereof.

[0027] In another embodiment, the nucleic acid(s) comprise a plasmid. In another embodiment, the nucleic acid(s) comprise an expression vector. In another embodiment, the expression vector is a lentivirus vector. In another embodiment, the nucleic acid is an RNA encoding the CEACAM5 binding agent.

[0028] In another embodiment, the nucleic acid further comprises a human somatostatin receptor 2 (SSTR2) coding sequence. In one embodiment, the nucleic acid encodes a full length SSTR2 polypeptide (aa 1-381; SEQ ID NO: 69), where the nucleic acid comprises a polynucleotide sequence set forth in SEQ ID NO: 71. In another embodiment, the nucleic acid encodes a truncated SSTR2 polypeptide (aa 1-381; SEQ ID NO: 70), where the nucleic acid comprises a polynucleotide sequence set forth in SEQ ID NO: 72. In a further embodiment, the nucleic acid encodes an SSTR2-CEACAM5 binding agent fusion protein comprising an amino acid cleavage sequence between the SSTR2 and CEACAM5 binding agent coding sequences. In one embodiment, the coding sequence of the CEACAM5 binding agent is 5’ of the SSTR2 coding sequence. In another embodiment, the SSTR2 coding sequence is 5’ of the CEACAM5 binding agent coding sequence. In another embodiment, the amino acid cleavage sequence is encoded by a self-cleaving 2A peptide sequence from porcine teschovirus- 1 (P2A), equine rhinitis A virus (E2A), thosea asigna virus (T2A), or foot-and- mouth disease virus (F2A), or a combination thereof. In a further embodiment, the amino acidcleavage sequence comprises an amino acid sequence set forth in any one of SEQ ID NOs: 73- 76.

[0029] In another aspect, the present disclosure provides population of immune cells comprising genetically engineered immune cells expressing or secreting any one of the CEACAM5 binding agents, and optionally one or more exogenous gene products disclosed herein. The genetically engineered immune cells may comprise any one of the nucleic acid or set of nucleic acids disclosed herein.

[0030] In another embodiment, the genetically engineered immune cells are T cells, natural killer (NK) cells, tumor infiltrating lymphocytes, dendritic cells, macrophages, neutrophils, eosinophils, basophils, mast cells, myeloid-derived suppressor cells, stem cells, precursors thereof, subtypes thereof, or a combination thereof; optionally wherein the immune cell is a human immune cell. In one embodiment, the genetically engineered immune cells are T cells or NK cells. In a further embodiment, the genetically engineered immune cells are further engineered to express an exogenous SSTR2 polypeptide.

[0031] In another embodiment, the population of immune cells further comprises a second population of genetically engineered immune cells. In one embodiment, the second population of genetically engineered immune cells is engineered to express an exogenous polypeptide of interest.

[0032] In another aspect, the present disclosure provides a method of treating cancer. In one embodiment, the method comprises administering to a subject in need thereof an effective amount of a CEACAM5 binding agent disclosed herein. In another embodiment, a cell therapy method of treating cancer comprises administering to a subject in need thereof an effective amount of a population of immune cells comprising genetically engineered immune cells expressing or secreting a CEACAM5 binding agent disclosed herein. In another embodiment, the subject is a human patient with cancer.

[0033] In another embodiment, the cancer is a solid tumor, such as a carcinoma, glioblastoma, or mesothelioma. In another embodiment, the carcinoma is a small cell lung carcinoma, non-small cell lung carcinoma, squamous cell lung carcinoma, large cell lung carcinoma, pancreatic carcinoma, pancreatic ductal carcinoma, prostate carcinoma, esophageal carcinoma, breast carcinoma, ovarian carcinoma, prostate carcinoma, colorectal carcinoma, bladder carcinoma, cervical carcinoma, hepatocellular carcinoma, renal hepatocellular carcinoma, gastric carcinoma, papillary carcinoma, adrenocortical carcinoma, pituitarycarcinoma, a head and neck carcinoma, an adenocarcinoma thereof, or a squamous cell carcinoma thereof. In another embodiment, the cancer is metastatic.

[0034] In another embodiment, the method of treating cancer further comprises administering to the subject a therapy to reduce tumor burden in the subject prior to administration of the CEACAM5 binding agent. In another embodiment, the method further comprises administering a therapy to a patient prior to, concurrently, or after a therapy against the cancer to reduce tumor burden, such as chemotherapy, immunotherapy, radiotherapy, and / or surgery. In another embodiment, prior to the cell therapy, the subject has been administered a lymphodepleting treatment to condition the subject for the cell therapy. In another embodiment, the lymphodepleting treatment comprises administering to the subject fludarabine and / or cyclophosphamide.

[0035] In another embodiment, the therapy comprises administering to the patient an immune checkpoint inhibitor, such as pembrolizumab (Keytruda™), ipilimumab (Yervoy™), nivolumab (Opdivo™), or atezolizumab (Tecentriq™). In another embodiment, the therapy comprises administration of a therapeutic antibody selected from the group consisting of abagovomab, adecatumumab, afutuzumab, alemtuzumab, altumomab, amatuximab, anatumomab, arcitumomab, bavituximab, bectumomab, bevacizumab, bivatuzumab, blinatumomab, brentuximab, cantuzumab, catumaxomab, cetuximab, citatuzumab, cixutumumab, clivatuzumab, conatumumab, daratumumab, drozitumab, duligotumab, dusigitumab, detumomab, dacetuzumab, dalotuzumab, ecromeximab, elotuzumab, ensituximab, ertumaxomab, etaracizumab, farietuzumab, ficlatuzumab, figitumumab, flanvotumab, futuximab, ganitumab, gemtuzumab, girentuximab, glembatumumab, ibritumomab, igovomab, imgatuzumab, indatuximab, inotuzumab, intetumumab, ipilimumab, iratumumab, labetuzumab, lexatumumab, lintuzumab, lorvotuzumab, lucatumumab, mapatumumab, matuzumab, milatuzumab, minretumomab, mitumomab, moxetumomab, namatumab, naptumomab, necitumumab, nimotuzumab, nofetumomab, ocaratuzumab, ofatumumab, obinutuzumab, olaratumab, onartuzumab, oportuzumab, oregovomab, panitumumab, parsatuzumab, patritumab, pemtumomab, pertuzumab, pintumomab, pritumumab, racotumomab, radretumab, rilotumumab, rituximab, robatumumab, satumomab, sibrotuzumab, siltuximab, simtuzumab, solitomab, tacatuzumab, taplitumomab, tenatumomab, teprotumumab, tigatuzumab, tositumomab, trastuzumab,tucotuzumab, ublituximab, veltuzumab, vorsetuzumab, votumumab, zalutumumab, CC49 and 3F8.

[0036] In another embodiment, the method of cell therapy further comprises administering to the patient a tyrosine kinase inhibitor capable of inhibiting TCR signaling, such as dasatinib, ponatinib, saracatinib, bosutinib, nilotinib, or a combination thereof. In a particular embodiment, the inhibitor is dasatinib. In another embodiment, the tyrosine kinase inhibitor is administered for a period of time sufficient to restore at least partial T cell function and then discontinued. In another embodiment, the tyrosine kinase inhibitor is administered continuously. In another embodiment, the tyrosine kinase inhibitor is administered intermittently. In another embodiment, the tyrosine kinase inhibitor is administered iteratively to facilitate periods of T cell inactivation (e.g., during pharmaceutical composition administration) and periods of T cell activation (e.g., during absence of pharmaceutical composition administration).

[0037] In another aspect, the present disclosure provides a method for treating a cancer and monitoring distribution of genetically engineered T cells or NK cells in a patient. The method comprises the steps of: (a) incubating the genetically engineered T cells or NK cells with a radioactive label that binds to SSTR2; (b) intravenously infusing the labeled T or NK cells into a patient in an amount of 104-108cells / kg patient; and detecting the labeled T or NK cell distribution by PET / CT imaging, wherein the labeled T or NK cells are infiltrated into cancer cells to kill the cancer cells. In another embodiment, the label is radioactively labeled DOTATOC or radioactively labeled DOTATATE. In a further embodiment, DOTATOC or DOTATATE is radiolabeled with68Ga. In some embodiments, the cancer is lung cancer, gastric cancer, pancreatic cancer, thyroid cancer, or breast cancer.

[0038] In another embodiment, a method for treating cancer and monitoring distribution of genetically engineered T cells or NK cells in a patient comprises the steps of: (a) intravenously infusing a population of genetically engineered T or NK cells into a patient, wherein the T or NK cells have been transduced to express at least 100,000 molecules of SSTR2 per cell; (b) injecting into the patient a radioactive label that binds to SSTR2 at least one hour prior to PET / CT imaging, and (c) detecting the labeled T or NK cell distribution by PET / CT imaging, wherein the labeled T or NK cells are infiltrated into cancer cells to kill thecancer cells. In some embodiments, the cancer is lung cancer, gastric cancer, pancreatic cancer, thyroid cancer, or breast cancer.

[0039] In another aspect, a method of producing a CEACAM5 binding agent disclosed herein comprises the steps of: introducing one or more expression vectors encoding the CEACAM5 binding agent; culturing the cells under conditions sufficient for expressing one or two polypeptide chains of the CEACAM5 binding agent; and harvesting the binding agent produced from the cultured cells. In another embodiment, the method further comprises the step of subjecting the one or two polypeptide chains to reducing conditions sufficient for forming disulfide bonds between the one or two polypeptide chains, thereby producing the binding agent.

[0040] In another aspect, a method of producing a population of genetically engineered immune cells disclosed herein comprises the step of introducing into a population of immune cells a nucleic acid or set of nucleic acids coding for any one of CEACAM5 binding agents disclosed herein, and optionally one or more exogenous nucleic acids for expressing one or more polypeptides of interest, to produce a population of genetically engineered cells expressing the CEACAM5 binding agent. In another embodiment, the immune cells include T cells, natural killer (NK) cells, tumor infiltrating lymphocytes, dendritic cells, macrographs, B cells, neutrophils, eosinophils, basophils, mast cells, myeloid-derived suppressor cells, stem cells, precursors thereof, or a combination thereof; optionally wherein the immune cell is a human immune cell. In a particular embodiment, the immune cells are T cells or NK cells. In another embodiment, the method further comprises the step of expanding the population of genetically engineered immune cells in the presence of IL-2 and / or a tyrosine kinase inhibitor capable of inhibiting TCR signaling. In another embodiment, the tyrosine kinase is dasatinib. In another embodiment, the tyrosine kinase is ponatinib, saracatinib, bosutinib, nilotinib, or a combination thereof.

[0041] The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will be apparent from the following drawings and detailed description of several embodiments, and also from the appended claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0042] FIG. 1 is a schematic depiction of a CAR expression construct used in the Examples described herein.

[0043] FIG. 2 depicts binding curves reflecting the binding of CEACAM5 to parent Affy58 CAR-T-cells and to two affinity -reduced Affy58 variant CAR-T cells, including the results of this analysis expressed in EC50 and fold change in binding relative to the parent Affy58 CAR-T cells.

[0044] FIG. 3 depicts binding curves reflecting the binding of CEACAM5 to parent Affy69 CAR-T-cells and to four affinity-reduced Affy69 variant CAR-T cells, including the results of this analysis expressed in EC50 and fold change in binding relative to the parent Affy69 CAR-T cells.

[0045] FIG. 4 depicts in vitro cytotoxicity of Affy58 parent CAR-T cells and two affinity -reduced variant Affy58 CAR-T cell populations against an LoVo Human Colon Adenocarcinoma Cell Line.

[0046] FIG. 5 depicts in vitro cytotoxicity of Affy58 parent CAR-T cells and two affinity -reduced variant Affy58 CAR-T cell populations against an HCC827 Human Lung Adenocarcinoma Cell Line.

[0047] FIG. 6 depicts in vitro cytotoxicity of Affy58 parent CAR-T cells and two affinity -reduced variant Affy69 CAR-T cell populations against an LoVo Human Colon Adenocarcinoma Cell Line.

[0048] FIG. 7 depicts in vitro cytotoxicity of Affy58 parent CAR-T cells and two affinity-reduced variant Affy69 CAR-T cell populations against an HCC827 Human Lung Adenocarcinoma Cell Line.

[0049] FIG. 8 shows the results of the antibody internalization evaluation described in Example 4 below.DETAILED DESCRIPTION OF THE INVENTION

[0050] The present disclosure is based on the unexpected discovery that an affinity tuned CEACAM5 binding domain with reduced affinity for CEACAM5 can be used to effectively kill CEACAM5 overexpressing cancer cells, while avoiding the undesirable effect of killing healthy cells and tissue.CEACAM5 Binding Molecules(A ) Reduced Affinity CEACAM5 Binding Domain

[0051] The CEACAM5 binding agent of the present disclosure comprises an affinity-tuned antigen binding domain with reduced affinity for the CEACAM5 protooncogene. The reduced affinity antigen binding domain is contained in or derived from an antibody and comprises a heavy chain variable (VH) region and a light chain variable (VL) region. Suitable VH and VL regions for inclusion in the CEACAM5 binding agent of the present disclosure are described in Table 8 (as determined following the antibody rules described by the Bioinformatics and Computational Biology group website at University College London).

[0052] The VH and VL regions herein are further subdivided into regions of hypervariability, also known as “complementarity determining regions” (“CDR”), interspersed with regions that are more conserved, which are known as “framework regions” (“FR”). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The extent of the framework region and CDRs can be precisely identified using methodology known in the art, for example, by the Kabat definition, the Chothia definition, the AbM definition, and / or the contact definition, all of which are well known in the art. See, e.g., Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, Chothia et al., (1989) Nature 342:877; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, Al-Lazikani et al (1997) J. Molec. Biol. 273:927-948; and Almagro, J. Mol. Recognit. 17:132-143 (2004). See also the Human Genome Mapping Project Resources at the Medical Research Council in the United Kingdom and the antibody rules described at the Bioinformatics and Computational Biology group website at University College London. Amino acid and nucleotide sequences corresponding to the VH, VL and CDR sequences are shown in Table 8. The CDR regions in Table 8 were identified through NCBI Blast.

[0053] As used herein, the amino acid positions of all constant regions and domains of the heavy and light chain are numbered according to the Kabat numbering system described in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991). Specifically, the Kabat numbering system (see pages 647-660 of Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)) is used for the light chain constant domain CL of kappa and lambda isotype and the Kabat EUindex numbering system (see pages 661-723) is used for the heavy chain constant domains (CHI, Hinge, CH2 and CH3).

[0054] In another embodiment, the VH and VL regions are “humanized.” Humanized variable regions (or antibodies) are derived from chimeric immunoglobulins, human immunoglobulins, immunoglobulin chains, or antigen-binding fragments thereof in which minimal sequences are derived from a non-human immunoglobulin. In another embodiment, the humanized variable regions (or antibodies) comprise residues from CDRs of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity, where the framework region (FR) residues from the non- human species are replaced by corresponding human residues. Furthermore, the humanized binding region may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences, but are included to further refine and optimize antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of human immunoglobulin consensus sequences.

[0055] In another embodiment, the CEACAM5 binding agent will further comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Methods for constructing humanized antibodies are also well known in the art. See, e.g., Queen et al., Proc. Natl. Acad. Sci. USA, 86:10029-10033 (1989).

[0056] In another embodiment, the EC50 of the CEACAM5 binding domain to its binding partner (e.g., CEACAM5) is greater than 5 pM, greater than 10 pM, greater than 15 pM, greater than 20 pM, greater than 25 pM, greater than 30 pM, greater than 35 pM, greater than 40 pM, greater than 50 pM, greater than 5 pM but less than 50 pM, or any range thereof. In another embodiment, the fold-change in EC50 relative to a corresponding wild-type CEACAM5 binding domain (from which the reduced affinity binding domain is derived) is greater than 500-fold, greater than 800-fold, greater than 1,000-fold, greater than 2,000-fold, greater than 5,000-fold, greater than 10,000-fold, greater than 20,000-fold, greater than 30,000- fold, greater than 800-fold but less than 30,000-fold, or any range thereof. In some embodiments, the EC50 value is derived from a CEACAM5 scFv or a chimeric artificial receptor (CAR) comprising an extracellular CEACAM5 binding domain.

[0057] In another embodiment, the CEACAM5 binding domain (e.g., CEACAM5 scFv) comprises a VH region with HC CDR1 and HC CDR2 regions comprising the amino acidsequences set forth SEQ ID NOs: 1 and 2, respectively, and an HC CDR3 region comprising an amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 4; and a VL region with LC CDR1, LC CDR2, and LC CDR3 regions comprising the amino acid sequences set forth in SEQ ID NOs: 5-7, respectively.

[0058] In another embodiment, the VH and VL region amino acid sequences are selected from the group consisting of: (a) SEQ ID NOs: 9 and 11, respectively; and (b) SEQ ID NOs: 10 and 11, respectively. In another embodiment, the CEACAM5 binding domain comprises an anti-CEACAM5 scFv comprising an amino acid sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 18.

[0059] In another embodiment, the CEACAM5 binding domain comprises VH and VL regions where: the HC CDR1 and HC CDR2 regions comprise the amino acid sequences set forth in SEQ ID NOs: 34 and 35, respectively, the HC CDR3 region comprises an amino acid sequence set forth in SEQ ID NO: 36 or 37; and the VL region comprises LC CDR1, LC CDR2, and LC CDR3 regions comprising the amino acid sequences set forth in SEQ ID NOs: 38-40, respectively.

[0060] In another embodiment, the CEACAM5 VH and VL region amino acid sequences are selected from SEQ ID NOs: 42 and 44, respectively; or SEQ ID NOs: 43 and 44, respectively. In some embodiments, the CEACAM5 binding domain comprises an anti- CEACAM5 scFv comprising an amino acid sequence set forth in SEQ ID NO: 50 or 51.

[0061] As used herein, the term “CEACAM5 binding agent” may be used interchangeably with the term “antibody.” An “antibody” as used herein is an immunoglobulin molecule capable of specific binding to a target protein, e.g., CEACAM5, through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule. As used herein, the term “antibody” encompasses not only intact (e.g., full-length) antibodies and heavy chain antibodies (e.g., an Alpaca heavy chain IgG antibody), but also antigen-binding fragments thereof (such as Fab, Fab', F(ab')2, Fv), single chain (scFv) antibodies, singledomain antibodies (sdAb; VHH), also known as nanobodies, mutants thereof, fusion proteins comprising an antibody portion, humanized antibodies, chimeric antibodies, diabodies, linear antibodies, single chain antibodies, multispecific antibodies (e.g., bispecific antibodies) and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. An antibody includes an antibody of any class, such as IgD, IgE, IgG, IgA, or IgM (or sub-class thereof),but need not be of any particular class. Depending on the antibody amino acid sequence of the constant domain of its heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0062] A CEACAM5 binding agent of the present disclosure may be derived from or include constant regions from any immunoglobulin isotype. Exemplary isotypes include but are not limited to either of the human IgGl, IgG2, IgG3, IgG4 isotypes, or a combination thereof.

[0063] Either of the human light chain constant regions, kappa, or lambda, may be included in the CEACAM5 binding agent. In another embodiment, both heavy chains of an antibody of the present invention are of an IgGl isotype, such as the IgGl kappa isotype.

[0064] In another embodiment, the CEACAM5 binding agent is a monospecific single chain polypeptide. In one embodiment, the monospecific single chain polypeptide is a CEACAM5-scFv. An scFv or the present disclosure comprises VH and VL regions connected by a peptide linker. In another embodiment, the scFv has a VI IA-VL orientation. In another embodiment, the scFv has a VI.A-VI I orientation. In another embodiment, the CEACAM5 binding domain is a CEACAM5 scFv comprising an amino acid sequence set forth in any one of SEQ ID NOs: 20, 21, 50, or 51. Peptide linkers used in scFv constructs are well known in the art and include, for example, the amino acid sequence (GxS)n in which x is an integer between 1-6, inclusive, and n is an integer between 1-10, inclusive. In another embodiment, the linker comprises the amino acid sequence (GGGS)n in which n is an integer between 1-10, inclusive (SEQ ID NO: 67). In one embodiment, the linker comprises the amino acid sequence multimer GGGGSGGGGSGGGGS (SEQ ID NO: 68).

[0065] In another embodiment, the monospecific single chain polypeptide is a CEACAM5 scFv fused to a monomeric immunoglobulin IgG constant region domain capable of pH-dependent binding to FcRn in order to provide extended in vivo half-life. Ying et al.,Front. Immunol., (2014), vol. 5, article 146. Exemplary monomeric Ig constant region domains include CH2, CH3, CH1-CH2, CH1-CH3, and CH1-CH2-CH3 polypeptide chains.(B) Bispecific and Multispecific CEACAM5 Binding Molecules

[0066] In another aspect, the present disclosure provides bispecific or multispecific CEACAM5 binding agents with enhanced T cell-mediated killing of CEACAM5-expressing tumor cells. The term “bispecific” means that the antibody is able to bind to at least two distinct antigenic determinants. A bispecific antibody comprises two antigen binding sites, each of which is specific for a different antigenic determinant. In certain aspects, the bispecific antibody is capable of simultaneously binding two antigenic determinants on the same cell or two antigenic determinants expressed on two distinct cells. As used herein, the term “bispecific” should be construed as covering bispecific and multispecific CEACAM5 binding agents (e.g., trispecific etc.).

[0067] A bispecific CEACAM5 binding agent according to the present disclosure may comprise a CEACAM5 binding domain described herein and a second antigen binding domain. Many different formats and uses of bispecific antigen binding agents are known in the art, and are reviewed by e.g., Kontermann; Drug Discov Today, 2015 Jul;20(7):838-47; Runcie et al., Mol. Med. (2018) 24:50; and Lou et al. (2022) 42:804-827. A bispecific or multispecific CEACAM5 binding agent according to the present invention is not limited to any particular bispecific or multispecific format or method of producing it.

[0068] In exemplary embodiments, the bispecific CEACAM5 binding agent is a bispecific scFv, bispecific CAR, a diabody, a tandem diabody, a DART, a minibody, an scFv- Fab, an scFv-scFab, a cross-over Fab, a Fab, an (scFv)2-Fab, a triabody, a tetrabody, or a VH- scFv.

[0069] In one embodiment, the bispecific CEACAM5 binding agent is a bispecific scFv comprising a second scFv having a different binding specificity. In some embodiments, within each antibody or antibody fragment (e.g., scFv) of a bispecific scFv, the VH can be upstream or downstream of the VL. In some embodiments, the upstream antibody or antibody fragment (e.g., scFv) is arranged with its VH (VHi) upstream of its VL (VLi) and the downstream antibody or antibody fragment (e.g., scFv) is arranged with its VL (VL2) upstream of its VH (VH2), such that the overall bispecific antibody molecule has the arrangement VHi- VL1-VL2-VH2. In other embodiments, the upstream antibody or antibody fragment (e.g., scFv) is arranged with its VL (VLi) upstream of its VH (VHi) and the downstream antibody orantibody fragment (e.g., scFv) is arranged with its VH (VH2) upstream of its VL (VL2), such that the overall bispecific antibody molecule has the arrangement VLi VH1-VH2-VL2. In some embodiments, a linker is disposed between the two antibodies or antibody fragments (e.g., scFvs), for example, between VLi and VL2 if the construct is arranged as VH1-VL1-VL2-VH2, or between VHi and VH2 if the construct is arranged as VL1-VH1-VH2-VL2. The linker may be a linker as described herein, e.g., a (Gly3-Ser)n or (Gly4-Ser)n linker, wherein n is 1 , 2, 3, 4, 5, or 6. In general, the linker between the two scFvs should be long enough to avoid mispairing between the domains of the two scFvs. In some embodiments, a linker is disposed between the VL and VH of the first scFv. In some embodiments, a linker is disposed between the VL and VH of the second scFv. In constructs that have multiple linkers, any two or more of the linkers may be the same or different. Accordingly, in some embodiments, the bispecific scFv comprises VLs, VHs, and may further comprise one or more linkers in an arrangement as described herein.

[0070] In another embodiment, the second antigen binding domain in the bispecific CEACAM5 binding agent has a binding specificity for a tumor associated antigen (TAA). In some embodiments, the second antigen binding domain targets a tumor- associated antigen (TAA), particularly one that is overexpressed in solid tumors and metastatic tumors. In certain embodiments, the second antigen binding domain is an scFv. Exemplary TAAs include, but are not limited to ICAM-1, EpCAM, CEACM5, EGFRvIII, Interleukin 3 receptor subunit a- spanning 4-domains Al (MS4A1 also known as CD20); CD22 molecule (CD22); CD229 molecule (CD229) CD24 molecule (CD24); CD248 molecule (CD248); CD276 molecule (CD276 or B7H3); CD3 molecule (CD3); CD33 molecule (CD33); CD38 molecule (CD38); CD44v6; CD5 molecule (CD5); CD56 molecule (CD56); CD7 molecule (CD7); CD70 molecule (CD70); CD72; CD79a; CD79b; TNF receptor superfamily member 8 (TNFRSF8 also known as CD30); KIT proto-oncogene receptor tyrosine kinase (CD117); V-set pre-B cell surrogate light chain 1 (VPREB1 or CD 179a); adhesion G protein-coupled receptor E5 (ADGRE5 or CD97); TNF receptor superfamily member 17 (TNFRSF17 also known as BCMA); SLAM family member 7 (SLAMF7 also known as CS1); LI cell adhesion molecule (LI CAM); C-type lectin domain family 12 member A (CLEC12A also known as CLL-1); tumor- specific variant of the epidermal growth factor receptor (EGFRvIII); thyroid stimulating hormone receptor (TSHR); Fms related tyrosine kinase 3 (FLT3); ganglioside GD3 (GD3); Tn antigen (Tn Ag); lymphocyte antigen 6 family member G6D (LY6G6D); Delta like canonical Notch ligand 3 (DLL3); Nectin cell adhesion molecule 4 (Nectin-4); Interleukin- 13 receptorsubunit alpha-2 (IL-13RA2); Interleukin 11 receptor subunit alpha (IL 1 IRA); mesothelin (MSLN); Receptor tyrosine kinase like orphan receptor 1 (R0R1); Prostate stem cell antigen (PSCA); erb-b2 receptor tyrosine kinase 2 (ERBB2 or Her2 / neu); Protease Serine 21 (PRSS21); Kinase insert domain receptor (KDR also known as VEGFR2); Lewis y antigen (LewisY); Solute carrier family 39 member 6 (SLC39A6); Fibroblast activation protein alpha (FAP); Hsp70 family chaperone (HSP70); Platelet-derived growth factor receptor beta (PDGFR-beta); Cholinergic receptor nicotinic alpha 2 subunit (CHRNA2); Stage-Specific Embryonic Antigen-4 (SSEA-4); Mucin 1, cell surface associated (MUC1); mucin 16, cell surface associated (MUC16); claudin 18 (CLDN18); claudin 6 (CLDN6); Epidermal Growth Factor Receptor (EGFR); Preferentially expressed antigen in melanoma (PRAME); Neural Cell Adhesion Molecule (NCAM); ADAM metallopeptidase domain 10 (ADAM 10); Folate receptor 1 (FOLR1); Folate receptor beta (FOLR2); Carbonic Anhydrase IX (CA9); Proteasome subunit beta 9 (PSMB9 or LMP2); Ephrin receptor A2 (EphA2); Tetraspanin 10 (TSPAN10); Fucosyl GM1 (Fuc-GMl); sialyl Lewis adhesion molecule (sLe); TGS5; high molecular weight-melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); tumor endothelial marker 7-related (TEM7R); G protein-coupled receptor class C group 5, member D (GPRC5D); chromosome X open reading frame 61 (CXORF61); ALK receptor tyrosine kinase (ALK); Polysialic acid; Placenta-specific 1 (PLAC1); hexasaccharide portion of globoH glycoceramide (GloboH); NY-BR-1 antigen; uroplakin 2 (UPK2); Hepatitis A virus cellular receptor 1 (HAVCR1); adrenoceptor beta 3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 family member K (LY6K); olfactory receptor family 51 subfamily E member 2 (OR51E2); TCR Gamma Alternate Reading Frame Protein (TARP); Wilms tumor protein (WT1); ETV6-AML1 fusion protein due to 12; 21 chromosomal translocation (ETV6-AML1); sperm autoantigenic protein 17 (SPA17); X Antigen Family, Member IE (XAGE1E); TEK receptor tyrosine kinase (Tie2); melanoma cancer testis antigen- 1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos- related antigen 1; p53 mutant; human Telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoints; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-Acetyl glucosaminyl-transferase V (NA 17); paired box protein Pax-3 (PAX3); Androgen receptor; Cyclin B 1; v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN); Ras Homolog Family Member C (RhoC); Cytochrome P450 IB 1 (CYP1B 1); CCCTC-Binding Factor (Zinc Finger Protein)-Like (BORIS); Squamous Cell Carcinoma Antigen Recognized By T Cells 3(SART3); Paired box protein Pax-5 (PAX5); proacrosin binding protein sp32 (OY-TES 1); lymphocyte- specific protein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2 (55X2); Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FC AR); Leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); Glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); and immunoglobulin lambda-like polypeptide 1 (IGLL1), TAG72, CD171, CAIX, Ephrin B2, Legumam, HPV E6 or E7, GFRa4, and combinations thereof.

[0071] Exemplary solid tumor-associated antigen targets for the second binding domain may include but are not limited to target antigens selected from ICAM-1, EpCAM, LFA-1, CEACAM5, SSTR2, mesothelin, MUC1, ROR1, B7H3, CDH3, CEA, DLL3, EGFR, GD2, o-acetyl-GD2, GPA33, GPC3, HER2, PSCA, PSMA, EGFRvIII, CS-1, Tn Ag, TAG72, CD44v6, KIT, IL-13Ra2, GD3, CD171, IL-IRa, VEGFR2, Lewis Y, CD24, PDGFR-beta, SSEA-4, folate receptor alpha, folate receptor beta, ERBB2, Her2 / neu, EGFR, NCAM, Ephrin B2, CAIX, LMP2, sLe, HMWMAA, TEM1 / CD248, TEM7R, FAP, Legumam, HPV E6 or E7, CLDN6, TSHR, GPRC5D, ALK, Ply sialic acid, PLAC1, globoH, NY-BR-1 , UPK2, HAVCR1, ADRB3, PANX3, GPR20, Ly6k, OR51E2, TARP, and GFRa4.

[0072] In another embodiment, the CEACAM5 binding agent is targeted to a solid tumor-associated antigen target selected from the group consisting of ICAM-1, EpCAM, LFA- 1, CEACAM5, SSTR2, mesothelin, MUC1, ROR1, B7H3, CDH3, CEA, DLL3, EGFR, GD2, GPA33, GPC3, HER2, PSCA, PSMA, and combinations thereof.

[0073] In one embodiment, the second antigen binding domain comprises an scFv binding to epithelial cell adhesion molecule (EpCAM), optionally comprising an amino acid sequence set forth in SEQ ID NO: 59. In another embodiment, the second antigen binding domain comprises an scFv binding to an aL subunit I domain of human lymphocyte function- associated antigen- 1 (LFA-1), optionally comprising an amino acid sequence set forth in SEQ ID NO: 78.

[0074] In some embodiments, the second antigen binding domain is a binding domain of a protein ligand. In one embodiment, the binding domain of a protein ligand comprises an ICAM-1 binding domain. In one embodiment, the ICAM-1 binding domain comprises the I domain of the aL subunit (e.g., SEQ ID NO: 78) of human lymphocyte function-associated antigen-1 (LFA-1), which is known to target ICAM-1. A wild-type (wt) Idomain encompasses amino acid residues 130-310 (SEQ ID NO: 78) of the 1145 amino acid long mature aL integrin subunit protein (SEQ ID NO: 77, which corresponds to amino acid residues 26-1170 of GenBank Accession No. NP_002200).

[0075] In some embodiments, the ICAM-1 binding domain comprises an I domain mutant disclosed in U.S. Pat. No. 10,428,136, which is incorporated herein by reference in its entirety. Such mutants differ in their affinity for ICAM-1. For example, I domain mutants having one mutation at F292A (Kd 20 pM), F292S (Kd 1.24 pM), L289G (Kd 196 nM), F265S (Kd 145 nM), and F292G (Kd 119 nM), or having two mutations at K287C / K294C (Kd 100 nM) in the wild-type I domain are suitable for the present invention. The above numbering of the amino acid residues is in reference to the amino acid sequence of the mature aL integrin of SEQ ID 77. Thus, in some embodiments, the I domain in the bispecific or multi- specific CEACAM5-CAR set forth in SEQ ID NO: 78 includes one mutation of F292A, F292S, L289G, F265S, or F292G, or with two mutations at K287C / K294C.In another embodiment, the protein ligand comprises an aL subunit I domain of human lymphocyte function-associated antigen- 1 (LFA-1) targeting ICAM-1. In another embodiment, the binding domain comprises an amino acid sequence set forth in SEQ ID NO: 78 or a mutant thereof.

[0076] In another embodiment, the bispecific CEACAM5 binding agent is an immune cell engager. In another embodiment, the immune cell engager is a bispecific T cell engager (BiTE). The T cell engagers contemplated herein include at least two binding domains: one that is able to bind, including simultaneously bind, to a T cell (typically via an antigenic determinant expressed on the T cell, such as CD3) and at least one antigen binding domain of a tumor-associated antigen, such as CEACAM5. As such, the binding domains in the BiTE join together a tumor cell and a T cell, thereby exerting its effects through cellular response(s) of T cells, particularly CD4+and CD8+T cells. The cellular responses include cytotoxic activity or lysis of target cells (e.g., CEACAM5 overexpressing tumor cells), cytokine secretion, cytotoxic effector molecule release, T cell activation, proliferation, differentiation, and / or expression of activation markers. In one embodiment, the bispecific T cell engager comprises a CEACAM5 binding domain, a CD3 binding domain, and optionally one or more additional antigen (e.g., TAA) binding domains.

[0077] In another embodiment, the bispecific CEACAM5 binding agent is a bispecific natural killer cell engager (NKCE). The NKCEs contemplated herein include at least two binding domains: one that is able to bind, including simultaneously bind, to a natural killer (NK) cell (typically via an antigenic determinant expressed on the NK cell, such as CD 16a)and at least one antigen binding domain of a tumor-associated antigen, such as CEACAM5. As such, the two binding domains join together a tumor cell and an NK cell, thereby exerting its effects through cellular responses of NK cells, particularly CD16+NK cells. The cellular responses include inducing or enhancing the activity of NK cells, particularly CD16+NK cells, such as cytotoxic activity or lysis of target cells (e.g., CEACAM5 overexpressing tumor cells), cytokine secretion, cytotoxic effector molecule release, proliferation, differentiation, and / or expression of activation markers. CD 16 (also known as FcyRIII) is a cell surface antigen expressed on certain immune cells, including NK cells. CD16 exists in a transmembrane form (CD16a, Fey receptor Illa), which is expressed e.g., on NK cells and activated macrophages, and in a glycosylphosphatidyl-inositol (GPI)-anchored form (CD 16b, FcyRIIIb), which is expressed on neutrophils. CD 16a binds tumor-bound IgGs to activate NK cells and facilitate antibody-dependent cell-mediated cytotoxicity (ADCC).

[0078] In another embodiment, the bispecific CEACAM5 binding agent is a bispecific macrophage engager (BiME) comprising a CEACAM5 binding domain and a second antigen binding domain for a protein expressed on macrophages, such as signal regulatory protein-a (SIRPa)-, CD47, CD89, and / or a Mer proto-oncogene tyrosine kinase (MerTK).

[0079] In another embodiment, the first and / or the second antigen binding domain is a Fab molecule. In another embodiment, the first antigen binding moiety is a crossover Fab molecule wherein either the variable or the constant regions of the Fab light chain and the Fab heavy chain are exchanged and the second antigen binding domain is a conventional Fab molecule.

[0080] In another embodiment, the bispecific CEACAM5 binding agent is in a one- chain format. In a further embodiment, such a binding agent comprises three peptide linkers (E1-E3, from N-terminus to C-terminus) separating the four variable regions in the bispecific binding agent. The three peptide linkers may be of the same length, or of different lengths. In some examples, the three peptide linkers are identical. In other examples, they are different in sequence and / or length. Non-limiting examples of such peptide linkers are provided elsewhere in the instant disclosure, for example, in SEQ ID NOs: 67 and 68. Optionally, the one-chain bispecific antibody may further comprise a fourth peptide linker at the C-terminus of the last variable region (from N~>C orientation) for connecting an Fc fragment.

[0081] In another embodiment, the CEACAM5 bispecific binding agent is in a two- chain format where each of the two polypeptides comprises a peptide linker separating the two variable regions therein. The peptide linkers in the two polypeptides may be of the same lengthand / or same sequence. Alternatively, they may differ in length, in sequence, or both. Nonlimiting examples of such peptide linkers are provided in e.g., SEQ ID NOs: 67 and 68.

[0082] In another embodiment, the bispecific CEACAM5 binding agent is in a two- chain format comprising a first polypeptide and a second polypeptide, each of which comprises one variable region of the first antigen-binding moiety and one variable region of the second antigen-binding moiety. In some examples, such a bispecific antibody may comprise: a first polypeptide comprising, from N-terminus to C-terminus, the first VL, a first peptide linker (LI), the second VH, and optionally a second peptide linker (L2); and a second polypeptide comprising, from N-terminus to C-terminus, the second VL, a third peptide linker (L3), and the first VH, and optionally a fourth peptide linker (L4).

[0083] In other embodiments, the bispecific antibody may comprise: a first polypeptide comprising, from N-terminus to C-terminus, the first VH, a first peptide linker (LI), the second VL, and optionally a second peptide linker (L2); and a second polypeptide comprising, from N-terminus to C-terminus, the second VH, a third peptide linker (L3), the first VL, and optionally a fourth peptide linker (L4). In some instances, the first polypeptide further comprises a first C-terminal fragment, which may be linked to the second VL via the L2 linker and the second polypeptide further comprises a second C-terminal fragment, which may be linked to the second VH via the L4 linker, such that the first C-terminal fragment and the second C-terminal fragment form a dimer.

[0084] In some embodiments, the first C-terminal fragment is a first Fc fragment of a first IgG molecule and the second C-terminal fragment is a second Fc fragment of a second IgG molecule, whereby the first Fc fragment and the second Fc fragment form an IgG Fc region. In some examples, the first Fc fragment comprises a first CH2 domain and a first CH3 domain, and the second Fc fragment comprises a second CH2 domain and a second CH3 domain. The first CH2 and the second CH2 domains may further comprise one or more amino acid modifications relative to a wild-type counterpart to form knob(s) and hole(s) to promote heterodimerization of the polypeptide chains as further described below. Alternatively, the first CH3 and the second CH3 domains each comprise one or more amino acid modification relative to a wild-type counterpart to form the knob(s) and hole(s).

[0085] In some embodiments, the bispecific CEACAM5 binding agents disclosed herein may comprise a C-terminal fragment to form a dimer. In some instances, the C-terminal fragment is an Fc fragment or a portion thereof e.g., comprising the CH2 domain, the CH3, domain, or both) from an immunoglobulin molecule. The C-terminal fragment may beobtained from any suitable Ig subfamily e.g., IgG, IgA, IgE, IgD, or IgM). In some examples, the C-terminal fragment may be an Fc fragment from an IgG (e.g., IgGl) such as a human IgG molecule.

[0086] When the bispecific binding agent is in the one-chain format, such a bispecific antibody may comprise the C-terminal fragment, which may be linked to the last variable region (in N~>C orientation) via a peptide linker comprising one or more cysteine residues enabling the formation of one or more disulfide bonds between two copies of the single-chain bispecific antibody to form a homodimer. In some instances, the linker may be the hinge domain of an Ig or mimic the hinge domain of an Ig.

[0087] When the bispecific binding agent is in the two-chain format, each polypeptide chain may comprise a C-terminal fragment, which can be linked to the C-terminal variable region via a peptide linker. In some examples, the C-terminal fragment may be the Fc fragment of an Ig, for example, IgG (e.g., human IgG). In some instances, the Fc fragments are of a naturally-occurring IgG molecule. In other instances, the Fc fragments may comprise one or more mutations to enhance heterodimer formation (between the two polypeptides of the bispecific antibody) and reduce or eliminate formation of homodimers (between two copies of one polypeptide of the bispecific antibody).

[0088] In some embodiments, the Fc fragment in the first polypeptide of the two- chain bispecific antibody and the Fc fragment in the second polypeptide of the bispecific antibody may comprise one or more knob / hole modifications in the CH2 domain, in the CH3 domain, or in both the CH2 and CH3 domains. Typically, the terms “a knob and a hole” or "knobs-into-holes" are used interchangeably herein. Knobs-into-holes amino acid changes is a rational design strategy known in the art for heterodimerization of the heavy (H) chains in the production of bispecific IgG antibodies. Carter, J. Immunol. Methods, 248(l-2):7-15 (2001), the relevant disclosures of which are incorporated by reference herein for the purpose and subject matter referenced herein.

[0089] In one embodiment, the "knobs-into-holes" provides an approach as described in, e.g., Ridgway JBB et al., (1996) Protein Engineering, 9(7): 617-21 and US 5,731,168, the relevant disclosures of each of which are incorporated by reference herein for the purpose and subject matter referenced herein. This approach has been shown to promote the formation of heterodimers of the first polypeptide and the second polypeptide chain, and hinder the assembly of corresponding homodimers. In one aspect, a knob is created byreplacing small amino side chains at the interface between CH3 domains with larger ones, whereas a hole is constructed by replacing large side chains with smaller ones.

[0090] In a specific embodiment, the "knob" mutation comprises T366W and the "hole" mutations comprise T366S, L368A and Y407V (Atwell S et al., (1997) J . Mol. Biol. 270: 26-35). In another specific embodiment, the "knob" mutations comprise T366W, S354C and the "hole" mutations comprise T366S, L368A, Y407V and Y349C, so that a disulfide bond is formed between the corresponding cysteine residues S354C and Y349C, further promoting heterodimer formation. Unless otherwise indicated, the Kabat numbering system is used in the present disclosure for describing positions of amino acid residues in an antibody molecule.

[0091] In some embodiments, coding regions of one or more functional domains in the CEACAM5 binding agents may be variants of their naturally occurring counterparts. In another embodiment, any of these coding regions may contain an amino acid sequence at least 80% (e.g., at least 85%, 90%, 95%, 98%, 99% or above) identical to its natural counterpart. As used herein, the “percent identity” of two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol. Biol. 215:403-10, 1990. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules of interest. Where gaps exist between two sequences, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0092] In some embodiments, the Fc region may contain up to 15 (e.g., up to 12, 10, 8, 6, 5, 4, 3, 2, or 1) amino acid residue substitutions relative to the wild-type counterpart. In some examples, the amino acid residue substitutions are conservative amino acid residue substitutions. As used herein, a “conservative amino acid substitution” refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to routine methods for altering polypeptide sequences known to those skilled in the art Conservative substitutions of amino acids may include substitutions within the following groups: ( (a) A A G, S; (b) R A K, H; (c) N A Q, H; (d) D A E, N; I C A S, A; (f) Q A N; (g) E A D, Q; (h) G A A; (i) H A N,Q; (j) I A L, V; (k) L A I, V; (1) K A R, H; (m) MA L, I, Y; (n) F A Y, M, L; (o) P A A; (p) S A T; (q) TA S; I W A Y, F; (s) Y A W, F; and (t) VA I, L.(C) Fc Domains

[0093] In some embodiments, the CEACAM5 binding agent comprises one or more immunoglobulin constant regions and / or an Fc domain. The Fc domain may be included to provide increased half-life, promote heterodimerization or homodimerization of antigen binding fragments, and confer enhanced binding to immunoglobulin Fc receptors associated with antibody-dependent cytotoxicity (ADCC), particularly when provided with one or more amino acid substitutions relative to its wild-type Fc domain counterpart.

[0094] In one embodiment, the CEACAM5 binding agent comprises an immunoglobulin constant region, such as an IgGl CHI domain, a CH2 region, a CH3 region, or a combination thereof. In another embodiment, the CEACAM5 binding agent comprises a single chain or double chain Fc domain. In another embodiment, the binding agent comprises an effectorless constant region or an effectorless Fc domain. In another embodiment, the binding agent comprises a mutant constant region or mutant Fc region genetically engineered to confer enhanced antibody dependent cellular cytotoxicity.

[0095] Fc receptors (FcR) constitute a family of immune cell surface proteins capable of binding to the Fc portion of antibodies. There are several types of Fc receptors, including Fey receptors, Fea receptors, Fes receptors, and neonatal Fc receptors (FcRn), which have different binding activities to IgG, IgA, IgE, and IgG antibodies, respectively. The Fey receptor subfamily includes FcyRI (CD64), FcyRIIA (CD32a), FcyRIIB (CD32b), Fey RUB (CD32c), FcyRIIIA (CD16a), and FcyRIIIB (CD16b). FcyRI has high binding affinity to IgGl and IgG3 antibodies, while the other FcyRs have low binding affinity to IgG antibodies.

[0096] The types of Fc receptors play different roles in the immune system. For example, FcyRIIIA receptors, which are expressed on NK cells and macrophages, can bind to antibodies that are attached to cancer cells, thereby triggering cancer cell death by antibodydependent cell-mediated cytotoxicity (ADCC). On the other hand, FcyRIIB receptors, expressed on B cells and dendritic cells, can down regulate the activity of the immune cells when binding to IgG antibodies.

[0097] As used herein, the term “Fc domain” or “Fc region” is used with reference to a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions.Although the boundaries of the Fc region of an IgG heavy chain might vary slightly, the human IgG heavy chain Fc region is usually defined to extend from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Therefore, an antibody produced by a host cell by expression of a specific nucleic acid molecule encoding a full- length heavy chain may include the full-length heavy chain, or it may include a cleaved variant of the full-length heavy chain. This may be the case where the final two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbering according to Kabat EU index). Therefore, the C-terminal lysine (Lys447), or the C-terminal glycine (Gly446) and lysine (K447), of the Fc region may or may not be present.

[0098] In another embodiment, the Fc domain comprises one or more amino acid substitutions that alter its binding to an Fc receptor and / or one or more of its effector functions. Typically, the variant Fc region includes the same one or more amino acid substitutions in each of the two subunits of the Fc domain. In one embodiment, the CEACAM5 binding agent comprises a variant Fc where its effector function for antibody-dependent cell-mediated cytotoxicity (ADCC) is enhanced to provide increased ADCC activity. For example, in one embodiment, the CEACAM5-binding agent is engineered to contain Fc domain variants having enhanced binding affinity and / or selectivity for FcyRIIIA (CD 16) relative to its wild-type parent Fc region in which mutations are introduced to produce the Fc variants. In another embodiment, the Fc variant has increased binding to FcyRIIIA and / or decreased binding to another Fc receptor, for example, FcyRIIB.

[0099] In another embodiment, the Fc variants used herein may have a binding affinity (KD) for FcyRIIIA of at least 10-5, 10-6, 10-7, 10-8, 10-9, IO-10M, or lower. An increased binding affinity corresponds to a decreased KD. Higher affinity binding of an Fc fragment for a first Fc receptor relative to a second Fc receptor can be indicated by a higher KA (or a smaller numerical value KD) for binding the first Fc receptor than the KA (or numerical value KD) for binding the second Fc receptor. In such cases, the Fc variant has specificity for the first Fc receptor relative to the second Fc receptor. In another embodiment, the Fc variants described herein have a higher binding affinity (a higher KA or smaller KD) to FcyRIIIA as compared to the binding affinity to FcyRIIB. Differences in binding affinity (e.g., forspecificity or other comparisons) can be at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1000, 10,000 or 100,000 fold, or any range thereof.

[0100] The Fc variants described herein may be designed by mutating one or more amino acid residues in the wild- type of human IgGl, IgG2, or IgG4 Fc fragments in light of the amino acid residues in the corresponding mouse IgG, for example, mouse IgGl. For example, in one embodiment, a variant Fc domain with increased binding affinity for FcyRIIIA and / or reduced affinity for FcyRIIB comprises, or consists essentially of, substitutions of two or three of the residues at positions 298, 333 and 334 of the Fc region, where the residues are substituted with alanine residues (i.e., S298A, E333A, and K334A). Alternatively, or in addition, the variant may include one or more mutations selected from T307A, T307Q, and A339T. In another embodiment, the one or more amino acid substitutions increase the binding affinity of the Fc domain to FcyRIII by at least 2-fold, at least 5-fold, or at least 10-fold. Alternatively, or in addition, the variant Fc may comprise one or more amino acid substitutions increasing the binding affinity of the Fc domain to FcRn.

[0101] In another embodiment, the Fc region in the CEACAM5 binding agent is engineered to exhibit substantially no binding to at least one Fc receptor selected from FcyRI (CD64), FcyRIIA (CD32a), FcyRIIB (CD32b), FcyRIIIA (CD16a), and FcyRIIIB (CD16b). In another embodiment, the variant Fc domain exhibits substantially no binding to any of the Fc receptors selected from FcyRI (CD64), FcyRIIA (CD32a), FcyRIIB (CD32b), FcyRIIIA (CD16a), and FcyRIIIB (CD16b). As used herein, “substantially no binding” refers to weak to no binding to a selected Fc gamma receptor or receptors. In another embodiment, “substantially no binding” refers to a reduction in binding affinity (i.e., increase in Kd) to an Fc gamma receptor of at least 1000-fold.

[0102] In another embodiment, the variant Fc domain has reduced or substantially no effector function activity. As used herein, “effector function activity” refers to antibody dependent cellular cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP) and / or complement dependent cytotoxicity (CDC). In another embodiment, the variant Fc domain exhibits reduced ADCC, ADCP or CDC activity, as compared to a wildtype Fc domain. In another embodiment, the variant Fc domain exhibits a reduction in ADCC, ADCP and CDC, as compared to a wildtype Fc domain. In another embodiment, an Fc domain exhibits substantially no effector function (i.e., the ability to stimulate or effect ADCC, ADCP or CDC).As used herein, “substantially no effector function” refers to a reduction in effector function activity of at least 1000-fold, as compared to a wildtype or reference Fc domain.

[0103] Exemplary amino acid mutations for use in the CEACAM5 binding agents of the present disclosure are described e.g., in Shields et al., J Biol Chem 9(2), 6591-6604 (2001); WO 99 / 58572; WO 2022226317; and U.S. Patent Nos. 6,737,056, 7,317,091, 7,355,008, and 11,186,648; and U.S. Patent Application No. 2022 / 0289803, the disclosures of which are expressly incorporated by reference herein.

[0104] Mutant Fc regions can be prepared by amino acid deletion, substitution, insertion, or modification using genetic or chemical methods well known in the art. Genetic methods may include site-specific mutagenesis of the encoding DNA sequence, PCR, gene synthesis, and the like. The correct nucleotide changes can be verified for example by sequencing.(D) Anti-CEACAM5 Drug Conjugates

[0105] In another aspect, the present disclosure provides an anti-CEACAM5 drug conjugate (used herein interchangeably with the term “antibody-drug conjugate”, “ADC” and “CEACAM5 ADC”) comprising a plurality of cytotoxic drugs attached a CEACAM5 binding agent by a plurality of flexible linkers. Thus, in one embodiment, the present disclosure provides CEACAM5 ADC comprising a cytotoxic drug, a linker, and a CEACAM5 binding agent disclosed herein. As used herein, the term "cytotoxic drug" refers to an agent that has a cytotoxic effect on a cell. A "cytotoxic effect" refers to the depletion, elimination and / or the killing of target cell, such as a cancer cell. Cytotoxic agents may include, for example, tubulin disrupting agents, topoisomerase inhibitors, DNA minor groove binders, and DNA alkylating agents.

[0106] The cytotoxic drug may be any agent known to inhibit the growth and / or replication of and / or kill cancer cells. The cytotoxic moiety used in anti-CEACAM5 drug conjugates should be of high stability in systemic circulation and in some cases, within cellular compartments such as lysosomes. Cytotoxic moieties for use in the methods and compositions of the present disclosure should have an in vitro IC50 in the nanomolar range, and even more preferably, in the sub-nanomolar range for cancer cell lines. In addition, a cytotoxic moiety used in the ADCs herein should be sufficiently soluble in the aqueous environment for administration. Other considerations for cytotoxic moieties for use in the ADCs include, but are not limited to low immunogenicity, small molecular weight, and long half-life. Thechemistry of the cytotoxic moiety should also allow for conjugation to the linker without interfering with the internalization or binding properties of the antibody or its anti-tumor effects.

[0107] Numerous agents having cytotoxic properties are known in the art. Any cytotoxic drug that can be modified to include a site of attachment to the CEACAM5 binding agent may be included in the CEACAM5 ADC for use in treating cancer according to the present disclosure. In general, they can be divided into two classes: microtubule disrupting agents and DNA- damaging agents. Non-limiting examples of classes of cytotoxic drugs belonging to these two classes include radionuclides, alkylating agents, topoisomerase I inhibitors, topoisomerase II inhibitors, DNA intercalating agents, RNA / DNA antimetabolites, cell cycle modulators, kinase inhibitors, protein synthesis inhibitors, histone deacetylase inhibitors, mitochondria inhibitors and antimitotic agents.

[0108] Microtubule disrupting agents are generally divided into two classes: auristatin and maytansinoids. Auristatin is a synthetic anti -neoplastic agent derived from dolastatin 10. Dolastin 10 is not suitable for use as part of an ADC due to its non-specific cytotoxic effects, but synthetic analogues of dolastin 10 such as monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF) are currently being used in ADC complexes. Both MMAE and MMAF work as cytotoxic drugs by blocking tubulin polymerization, resulting in cell cycle arrest and apoptosis.

[0109] Maytansinoids are a second class of microtubule disrupting agent that can be used as the cytotoxic moiety in the CEACAM5 ADC. Maytansinoids are isolated from the maytansine, a benzoansamacrolide. These drugs inhibit tubulin polymerization resulting in mitotic arrest and cell death. Derivatives of maytansine, mertansine (DM1) and ravtasine (DM4) have both been used as the cytotoxic moiety in ADCs.

[0110] DNA-damaging agents can also be used as the cytotoxic moiety in the CEACAM5 ADC. DNA-damaging agents can be divided up into three main classes including calicheamicin, duocarmycin, and doxorubicin. Calicheamicins are a class of enediyne antitumor antibiotics derived from Micromonospora echinospora. Calicheamicin binds to the minor groove of DNA and stops DNA replication causing cell cycle arrest and cell death. Duocarmycin is a derivative extracted from Streptomyces strains of bacteria. Duocarmycin are another example of agents that bind to the minor groove of DNA and causes irreparable DNA alkylation, disrupting the nucleic acid architecture and structural integrity. Doxorubicin is a DNA intercalating agent that inhibits DNA synthesis, resulting in cell death.Pyrrolobenzodiazepines (PBDs) and their derivatives constitute another class of DNA- damaging agents. PBDs translocate to the nucleus where they crosslink DNA, preventing replication during mitosis, damaging DNA by inducing single strand breaks, and subsequently leading to apoptosis. Some PBDs also have the ability to recognize and bind to specific sequences of DNA.

[0111] In one embodiment, the CEACAM5 ADC comprises a cytotoxic drug selected from the group consisting of a maytansinoid, a camptothecin, a dolastatin, an aurastatin, a pyrrolobenzodiazepine (PBD), a calicheamicin, a duocarmycin, a tubulolysin, or an analogue thereof. In another embodiment, the cytotoxic drug is selected from the group consisting of deruxtecan, exatecan, SN-38, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAE), Auristatin F-hydroxypropylamide, AurOlOl, emtansine (DM1), Maytansinoid DM4, duocarmycin, and duostatin-5.

[0112] One of skill in the art can pair specific classes of cytotoxic drugs with certain types of tumors. As a non-limiting example, one of skill in the art may choose particularly effective cytotoxic drugs from the DNA intercalating agent class or even a specific DNA intercalating agent if the targeted tumor has exhibited properties of multi-drug resistance.

[0113] Also included in a CEACAM5 ADC is a linker connecting the cytotoxic drug to the CEACAM5 binding agent in the ADC. The linker may be short, long, hydrophobic, hydrophilic, straight, flexible, or rigid, or may have segments comprised of one or more of the preceding properties. The linker may be linked to one or more sites on the binding agent or antibody. Such linkages may be covalent. As can be appreciated by one of skill in the art, the linker can be directly covalently linked to the antibody and the cytotoxic drug or there can be a spacer between the linker and the antibody and / or a spacer between the linker and the cytotoxic drug. Linkers that are useful in ADCs need to be stable while in systemic circulation to avoid the release of the cytotoxic drug in order to minimize off-target effects. However, the linkers should possess the quality of being able to unleash the cytotoxic drug upon internalization into the target tumor cell after antibody binding. Linkers can be divided into cleavable and non-cleavage linkers.

[0114] Non-cleavable linkers consist of stable bonds that resist proteolytic degradation and provide higher stability than cleavable linkers. For noncleavable ADC linkers, the release of drug does not depend on the differential properties between the plasma and some cytoplasmic compartments. The mechanism of action of non-cleavable linkers is based on the internalization of the ADC after the antibody has bound to its target on the surface of the targettumor cell. After internalization, the ADC complex is then degraded in the lysosome of the target cell that results in the release of the cytotoxic drug inside the target cell to facilitate the tumor cell killing effects of the ADC. Because the non-cleavable linker is more stable, it minimizes the chances of releasing the cytotoxic drug at off-target sites and thus do not harm healthy cells or minimizing off-target effects. In one embodiment, the CEACAM5 ADC comprises a non-cleavable linker selected from the group consisting of maleimidocaproyl (MC), succinimidyl-4-(N-maleimidomethyl)cyclohexane-l -carboxylate (SMCC), and thioether-containing linker. In another embodiment, the CEACAM5 ADC comprises an SMCC linker and a microtubule-disrupting agent, such as DM1 (emtansine).

[0115] Cleavable linkers are the second major class of linkers used as part of an ADC complex. The main feature of cleavage linkers is that they are sensitive and are cleaved as a result of environmental factors such as pH, specific lysosomal enzymes, etc. Different classes of cleavable linkers include but are not limited to acid- sensitive or acid-labile linkers, lysosomal protease-sensitive linkers, beta-glucuronide liners, and glutathione- sensitive disulfide linkers. Acid sensitive or acid-labile linkers are linkers that are stable at certain pH, usually at alkaline pH such as in systemic circulation and are sensitive to acidic environments such as lysosomal and / or endosomal acidic tumor microenvironments. Upon internalization within the target tumor cells, the linker gets hydrolyzed in its pH sensitive environment. Lysosomal protease- sensitive linkers are also known as peptide-based linkers. These linkers take advantage of the higher expression of some lysosomal proteases in tumor cells compared to non-tumor cells such as cathepsin B.

[0116] Lysosomal protease- sensitive linkers such as cathepsin B sensitive linkers are selectively taken up into tumor cells through receptor-mediated endocytosis. Another advantage of lysosomal protease-sensitive linkers is their stability in unsuitable pH environments and resistance to serum enzymes making them stable in systemic circulation and decreasing off target effects. An example of a lysosomal protease sensitive linker is valinecitrulline (v-c). In one embodiment, the CEACAM5 ADC comprises a cleavable linker, such as maleimidocaproyl-L-valine-L-citrulline-p-aminobenzoyl carbamate (mc-VC-PABC) or maleimido-dPEG8-L-valine-L-alaline-p-aminobenzoyl carbamate (M-dPEG8-VA-PABC).

[0117] Beta- glucuronide linkers are another class of protease- sensitive linker that is recognized by and hydrolyzed by beta-glucuronidase for the cytotoxic drug release from the ADC complex. Lysosomes and tumor necrotic regions are high in beta-glucuronidase, the enzyme being inactive at physiologic pH and active at lysosomal pH. This selectivity allowsfor cleavage of the linker and thereby the release of the cytotoxic moiety at desired site and minimizing off target effects.

[0118] Glutathione-sensitive disulfide linkers are another class of linkers that can be used in the CEACAM5 ADCs described herein. Glutathione is a low molecular weight thiol that is found in intracellular compartments of cells. Glutathione is highly released during cell survival, tumor growth and cell stress conditions such as hypoxia, therefore higher concentrations of glutathione can be found in tumors compared to non-tumor tissues Glutathione-sensitive linkers are stable in systemic circulation and are selectively cleaved when targeted to the tumor by the ADC by the higher concentration of glutathione.

[0119] In another embodiment, the enzymatically cleavable ADC linkers includes a self-immolative spacer to spatially separate the drug from the site of enzymatic cleavage. The direct attachment of a drug to a peptide ADC linker can result in proteolytic release of an amino acid adduct of the drug, thereby impairing its activity. The use of a self-immolative spacer allows for the elimination of the fully active, chemically unmodified drug upon amide bond hydrolysis. One self-immolative spacer is the bifunctional para-aminobenzyl alcohol group, which is linked to the peptide through the amino group, forming an amide bond, while amine containing drugs can be attached through carbamate functionalities to the benzylic hydroxyl group of the ADC linker (PABC). The resulting prodrugs are activated upon protease- mediated cleavage, leading to a 1,6-elimination reaction releasing the unmodified drug, carbon dioxide, and remnants of the ADC linker group.

[0120] The linkers may be chemically stable to conditions outside the cell but designed to cleave, degrade, and / or otherwise detach once inside the cell. Other ADCs need not be internalized by the targeted tumor cell and can achieve their cytotoxic effects on the surface of the tumor cell after the antibody of the ADC complex has bound to its epitope. A wide variety of linkers useful for linking cytotoxic drugs are known in the art, which may be used to link cytotoxic drugs to the CEACAM5 binding agents of the present disclosure to form CEACAM5 ADCs for treating cancer as disclosed herein.

[0121] The number of cytotoxic drugs linked to the CEACAM5 binding moiety of the ADC (drug-to-antibody ratio: DAR) can vary and may be limited only by the number of available attachments sites on the antigen binding moiety and the number of agents linked to a single linker. In another embodiment, the CEACAM5 ADC described herein may have a DARin the range of about 1-16, 1-14, 1-12, 1-10, 2-10, 2-8, 2-6, 2-4, 3-5, 6-8, 8-16, 2, 4, 6, 8, 10, 12, 14, or 16.

[0122] The DAR value can vary with the nature of the antibody and the drug used along with the experimental conditions used for the conjugation (DAR, reaction time, nature of the solvents and / or cosolvents). Thus, the contact between the antibody and the drug may lead to a mixture comprising several conjugates differing from one another by different drug- to-antibody ratios and may further include free antibodies and / or aggregates. The DAR that is determined is thus a mean value. DARs may be analyzed by UV spectrometry, monomer content may be analyzed by SEC-HPLC, and free drug content may be analyzed by RP-HPLC.

[0123] In another embodiment, the linker may link a single cytotoxic drug to the antigen binding moiety of the CEACAM5 ADC. In certain embodiments where the CEACAM5 ADC includes more than one cytotoxic drug, each agent may be the same or different. Further, as long as the CEACAM5 ADC does not exhibit unacceptable levels of aggregation under the conditions of use and / or storage, CEACAM5 ADCs with DARs of twenty, or even higher, are contemplated.II. Methods for Producing CEACAM5 Binding Agents, CEACAM5 Expressing Immune Cells, and CEACAM5 ADCs

[0124] The CEACAM5 binding agents of the present disclosure include affinity- reduced CEACAM5 binding agents comprising a heavy chain variable (VH) region and a light chain variable (VL). In another embodiment, nucleic acids encoding the CEACAM5 binding agents are prepared from nucleic acids encoding parent anti-CEACAM5 antibodies that have been subjected to site-directed mutagenesis of one or more CDRs in the VH and / or VL regions as exemplified in Example 1. Additional binding domains for other antigen targets may be prepared using polynucleotide sequences disclosed in the art or cloned from suitable nucleic acid sources.

[0125] In an alternative, the polynucleotide sequence may be used for genetic manipulation to, e.g., humanize the antibody or to improve the affinity (affinity maturation), or other characteristics of the antibody. For example, the constant region may be engineered to more resemble human constant regions to avoid immune response if the antibody is from a non-human source and is to be used in clinical trials and treatments in humans. Alternatively, or in addition, it may be desirable to genetically manipulate the antibody sequence to obtain lower affinity to the target antigen and greater efficacy in enhancing the activity of CEACAM5.It will be apparent to one of skill in the art that one or more polynucleotide changes can be made to the antibody and still maintain its binding specificity to the target antigen.

[0126] Genetically engineered antibodies, such as humanized antibodies, chimeric antibodies, single-chain antibodies, and bi-specific antibodies, can be produced via, e.g., conventional recombinant technology. In one example, DNA encoding a monoclonal antibody specific to a target antigen can be readily isolated and optionally sequenced using conventional procedures e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the monoclonal antibodies). Once isolated, the DNA fragments may be inserted into one or more expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells to produce antibodies in the recombinant host cells. See, e.g., PCT Publication No. WO 87 / 04462. The DNA can then be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences, Morrison et al., (1984) Proc. Nat. Acad. Sci. 81:6851, or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a nonimmunoglobulin polypeptide. In that manner, genetically engineered antibodies, such as “chimeric” or “hybrid” antibodies; can be prepared that have the binding specificity of a target antigen.

[0127] Methods for constructing humanized antibodies are also well known in the art. See, e.g., Queen et al., Proc. Natl. Acad. Sci. USA, 86:10029-10033 (1989). In one example, variable regions of VH and VL of a parent non-human antibody are subjected to three- dimensional molecular modeling analysis following methods known in the art. Next, framework amino acid residues predicted to be important for the formation of the correct CDR structures are identified using the same molecular modeling analysis. In parallel, human VH and VL chains having amino acid sequences that are homologous to those of the parent non- human antibody are identified from any antibody gene database using the parent VH and VL sequences as search queries. Human VH and VL acceptor genes are then selected. The CDR regions within the selected human acceptor genes can be replaced with the CDR regions from the parent non-human antibody or functional variants thereof. When necessary, residues within the framework regions of the parent chain that are predicted to be important in interacting withthe CDR regions (see above description) can be used to substitute for the corresponding residues in the human acceptor genes.

[0128] A single-chain antibody can be prepared via recombinant technology by linking a nucleotide sequence coding for a heavy chain variable region and a nucleotide sequence coding for a light chain variable region. Preferably, a flexible linker is incorporated between the two variable regions. Alternatively, techniques described for the production of single chain antibodies (U.S. Patent Nos. 4,946,778 and 4,704,692) can be adapted to produce a phage-display, yeast-display, mammalian cell-display, or mRNA-display scFv library and scFv clones specific to CEACAM5 can be identified from the library following routine procedures.

[0129] In an exemplary embodiment, nucleic acids encoding the VH and VL sequences (e.g., scFvs) or heavy and light chains of an anti-CEACAM5 antibody or a bispecific antibody as described herein can be cloned into one or more expression vectors. Expression vectors for stable or transient expression of the nucleic acids encoding the CEACAM5 binding agent of present disclosure can be used for preparative synthesis of the CEACAM5 binding agents from suitable cell sources, including bacteria, yeast, insect, or mammalian cells, or to prepare immune cells (e.g., T or NK cells) genetically engineered to express or secrete the CEACAM5 binding agents for adoptive cell therapy. For example, nucleic acids encoding the CEACAM5 binding agents may be cloned into a suitable expression vector, such as a viral vector in operable linkage to a suitable promoter. Expression constructs may be prepared using standard recombinant DNA techniques well known to those skilled in the art. In some instances, the selection of expression vectors / plasmids / viral vectors may depend on the type of host cells for expression of the CEACAM5 binding agent and the extent to which they are suitable for integration and replication in eukaryotic cells.

[0130] In one example, each nucleotide sequence encoding the heavy and / or light chains in an antibody is in operable linkage to a distinct promoter. Alternatively, the nucleotide sequences encoding the heavy chain and the light chain can be in operable linkage with a single promoter, such that both heavy and light chains are expressed from the same promoter. In such instances, an internal ribosomal entry site (IRES) or a P2A element can be inserted between the heavy chain and light chain encoding sequences. In another example, the nucleotide sequences encoding heavy and light chains of the antibody are cloned into two vectors, which can be introduced into the same or different cells. When the two chains are expressed in different cells, each of them can be isolated from the host cells expressing such and the isolatedheavy chains and light chains can be mixed and incubated under suitable conditions allowing for the formation of the antibody.

[0131] In another embodiment, methods for preparing an antibody described herein involve a recombinant expression vector that encodes both the heavy chain and the light chain of a CEACAM5 binding agent, or encodes both chains of a two-chain bispecific antibody as also described herein. The recombinant expression vector can be introduced into a suitable host cell (e.g., a dhfr- CHO cell) by a conventional method, e.g., calcium phosphate-mediated transfection. Positive transformant host cells can be selected and cultured under suitable conditions allowing for the expression of the two polypeptide chains that form the antibody, which can be recovered from the cells or from the culture medium. When necessary, the two chains recovered from the host cells can be incubated under suitable conditions allowing for the formation of the antibody.

[0132] In one example, two recombinant expression vectors are provided, one encoding the heavy chain of the anti-CEACAM5 binding agent or one of the two chains of a two-chain bispecific antibody disclosed herein and the other encoding the light chain of the anti-CEACAM5 antibody or the other chain of the bispecific antibody. Both of the two recombinant expression vectors can be introduced into a suitable host cell (e.g., dhfr- CHO cell) by a conventional method, e.g., calcium phosphate-mediated transfection. Alternatively, each of the expression vectors can be introduced into a suitable host cell. Positive transformants can be selected and cultured under suitable conditions allowing for the expression of the polypeptide chains of the antibody. When the two expression vectors are introduced into the same host cells, the antibody produced therein can be recovered from the host cells or from the culture medium. If necessary, the polypeptide chains can be recovered from the host cells or from the culture medium and then incubated under suitable conditions allowing for formation of the antibody. When the two expression vectors are introduced into different host cells, each of them can be recovered from the corresponding host cells or from the corresponding culture media. The two polypeptide chains can then be incubated under suitable conditions for formation of the antibody.

[0133] In certain embodiments, antigen-binding fragments of an intact antibody (full-length antibody) can be prepared via routine methods. For example, F(ab')2 fragments can be produced by pepsin digestion of an antibody molecule, and Fab fragments that can be generated by reducing the disulfide bridges of F(ab')2 fragments. Methods for makingbispecific antibodies are well known in the art and can be made using standard recombinant DNA technologies. See e.g., Brinkmann, U. et al. (2017) MABS:9(2): 182-212.

[0134] In certain embodiments, a retroviral vector (e.g., lentivirus-based vector) can be used to introduce the exogenous nucleic acid(s) into the immune cells disclosed herein. Preferably, the retroviruses (RVs) are replication-defective (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Retrovirus expression systems are well established and known to those skilled in the art for high-efficiency transduction of genes in vitro, ex vivo, and in vivo. Standard protocols for producing replication-defective RVs (including the steps of incorporating exogenous genetic material into retroviral plasmid vectors, transfecting and encapsidating retroviral plasmid vectors using retroviral packaging cell lines to produce recombinant RV particles, collecting recombinant RV particles from tissue culture media, and infecting target cells with the recombinant RV particles) are known in the art. Lentivirus vectors are particularly useful for producing engineered immune cells in view of the natural tropism of lentiviruses for immune cells. In another embodiment, a lentivirus vector is used for delivering the nucleic acids encoding the CEACAM5 binding agents (and optionally other gene products) into immune cells, such as T cells and NK cells.

[0135] A variety of promoters can be used for expression of the CEACAM5 binding agents described herein, including, but not limited to, cytomegalovirus (CMV) intermediate early promoter, a viral LTR such as the Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, the simian virus 40 (SV40) early promoter, E. coli lac UV5 promoter, and the herpes simplex virus (HSV)-thymidine kinase (TK) promoter.

[0136] Regulatable promoters can also be used. Such regulatable promoters include those using the lac repressor from E. coli as a transcription modulator to regulate transcription from lac operator-bearing mammalian cell promoters [Brown, M. et al., Cell, 49:603-612 (1987)], those using the tetracycline repressor (tetR) [Gossen, M., and Bujard, H., Proc. Natl. Acad. Sci. USA 89:5547-5551 (1992); Yao, F. et al., Human Gene Therapy, 9:1939- 1950 (1998); Shockelt, P„ et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)]. Other systems include FK506 dimer, VP 16 or p65 using estradiol, RU486, diphenol murislerone, or rapamycin. Inducible systems are available from Invitrogen, Clontech and Ariad.

[0137] Regulatable promoters that include a repressor with the operon can be used. In one embodiment, the lac repressor from E. coli can function as a transcriptional modulator to regulate transcription from lac operator-bearing mammalian cell promoters [M. Brown etal., Cell, 49:603-612 (1987); Gossen and Bujard (1992); M. Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992)] combined the tetracycline repressor (tetR) with the transcription activator (VP 16) to create a tetR- mammalian cell transcription activator fusion protein, tTa (tetR- VP 16), with the tetO-bearing minimal promoter derived from the human cytomegalovirus (hCMV) major immediate-early promoter to create a tetR-tet operator system to control gene expression in mammalian cells. In one embodiment, a tetracycline inducible switch is used. The tetracycline repressor (tetR) alone, rather than the tetR-mammalian cell transcription factor fusion derivatives can function as potent trans-modulator to regulate gene expression in mammalian cells when the tetracycline operator is properly positioned downstream for the TATA element of the CMVIE promoter (Y ao et al., Human Gene Therapy, 10(16): 1392-1399 (2003)). One particular advantage of this tetracycline inducible switch is that it does not require the use of a tetracycline repressor- mammalian cells transactivator or repressor fusion protein, which in some instances can be toxic to cells (Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992); Shockett et al., Proc. Natl. Acad. Sci. USA, 92:6522- 6526 (1995)), to achieve its regulatable effects.

[0138] Additionally, the vector can contain, for example, some or all of the following: a selectable marker gene, such as the neomycin gene for selection of stable or transient transfectants in mammalian cells; enhancer / promoter sequences from the immediate early gene of human CMV for high levels of transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; SV40 polyoma origins of replication and ColEl for proper episomal replication; internal ribosome binding sites (IRESes), versatile multiple cloning sites; and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA. Suitable vectors and methods for producing vectors containing transgenes are well known and available in the art.

[0139] Examples of polyadenylation signals useful to practice the methods described herein include, but are not limited to, human collagen I polyadenylation signal, human collagen II polyadenylation signal, and SV40 polyadenylation signal.

[0140] One or more vectors (e.g., expression vectors) comprising nucleic acids encoding any of the antibodies may be introduced into suitable host cells for producing the CEACAM5 antibodies (or binding agents). The host cells can be cultured under suitable conditions for expression of the antibody or any polypeptide chain thereof. Such antibodies or polypeptide chains thereof can be recovered by the cultured cells (e.g.. from the cells or the culture supernatant) via a conventional method, e.g., affinity purification. If necessary,polypeptide chains of the antibody can be incubated under suitable conditions for a suitable period of time allowing for production of the antibody.

[0141] Standard molecular biology techniques are used to prepare the recombinant expression vector, viral particles, transfect the host cells, select for transformants, culture the host cells and recovery of the antibodies from the culture medium. For example, some antibodies can be isolated by affinity chromatography with a Protein A or Protein G coupled matrix. Any of the nucleic acids encoding the heavy chain, the light chain, or both of an anti- CEACAM5 antibody or any of the bispecific antibodies as described herein, vectors (e.g., expression vectors) containing such; and host cells comprising the vectors are within the scope of the present disclosure.

[0142] To construct genetically engineered immune cells (e.g., T and NK cells) expressing the CEACAM5 binding agents described herein (as disclosed, for example, in Table 8), a starting population of parent immune cells is obtained from any source, such as peripheral blood mononuclear cells (PBMCs), bone marrow, or tissues such as spleen, lymph node, thymus, stem cells, or tumor tissue. Sources suitable for obtaining T or NK cells are well known to one of skill in the art. In another embodiment, the population of immune cells is derived from PBMCs. The T cells or NK cells may be expanded within the population of cells obtained by co-incubating the cells with stimulatory molecules. As a non-limiting example, anti-CD3 and anti-CD28 antibodies may be used for expansion of T cells. In another embodiment, the T cells, or NK cells are enriched from the immune cell population. Such enriched cell subpopulations may be expanded and / or activated in vitro prior to genetically engineering the cells through introduction of an expression vector or cassette expressing the CEACAM5 binding agent as described above.

[0143] The CEACAM5 ADCs of the present disclosure may be synthesized using methods known in the art. The specific chemistry of linking the antibody to the linker and also to the cytotoxic drug will depend upon, among other things, the identity of the cytotoxic drug, the linker and any spacer that is attached to the antibody. In another embodiment, each linker is attached to the binding agent via an interchain disulfide residue, a lysine residue, an engineered cysteine residue, a glycan, a modified glycan, an N-terminal residue of the binding agent or a polyhistidine residue attached to the binding agent. Generally, the chemistry used to link the linker to the antibody and the cytotoxic drug should not alter the integrity of the antigen binding domain so as to alter its ability to bind to its target. Preferably, the binding properties of the ADC will closely resemble those of the unconjugated antibody. A variety ofchemistries and techniques for conjugating molecules to biological molecules such as antibodies are well-known in the art. See, e.g., Alley et al., Current Opinion in Chemical Biology 2010 14:1-9; Senter, Cancer J., 2008, 14(3): 154-169.

[0144] In another embodiment, a linker is first attached to the cytotoxic drug, followed by the cytotoxic drug-linker being attached to the binding agent. In another embodiment, the linker is first attached to the binding agent, followed by the binding agentlinker being attached to the cytotoxic drug. The skilled artisan will appreciate that the selected attachment method can be determined according to linker and the cytotoxic drug. In another embodiment, the cytotoxic drug is attached to CEACAM5 binding agent via a linker in a manner that reduces the activity of the cytotoxic drug until it is released from the conjugate (e.g., by hydrolysis, by proteolytic degradation or by a cleaving agent.).

[0145] The CEACAM5 ADC may be prepared by several routes employing organic chemistry reactions, conditions, and reagents known to those skilled in the art, including: (1) reaction of a nucleophilic group on the antibody (or binding agent) with a bifunctional crosslinking reagent to form an antibody-linker intermediate via a covalent bond, followed by reaction with the cytotoxic drug; and (2) reaction of a nucleophilic group of a drug (e.g., a cytotoxic drug) with the bifunctional crosslinking reagent to form a drug-linker via a covalent bond, followed by reaction with a nucleophilic group in the antibody or binding agent.

[0146] As used herein, a “bifunctional crosslinking reagent” refers to a reagent that possesses two reactive groups: one is capable of reacting with the antibody (or binding agent), while the other is capable of reacting with the cytotoxic drug to link the antibody with the cytotoxic drug, thereby forming an ADC. Any suitable bifunctional crosslinking reagent can be used in connection with the present disclosure, so long as the linker reagent provides for retention of the drug, e.g., cytotoxicity, and targeting characteristics of the antibody. Preferably, the linker molecule joins the drug to the antibody or binding agent through chemical bonds, such that the drug and antibody are chemically coupled e.g., covalently bonded) to each other.

[0147] Bifunctional crosslinking reagents include, but are not limited to, N- succinimidyl-3-(2-pyridyldithio)butyrate (SPDB), N-succinimidyl-3-(2- pyridyldithio)propionate (SPDP), sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane- 1- carboxylate (Sulfo-SMCC), N-succinimidyl-4-(maleimidomethyl)cyclohexanecarboxylate (SMCC), N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-l-carboxy-(6-amidocaproate) (LC-SMCC), 4-maleimidobutyric acid N-hydroxysuccinimide ester (GMBS), 3-maleimidocaproic acid N-hydroxysuccinimide ester (EMCS), m-maleimidobenzoyl-N- hydroxysuccinimide ester (MBS), N-(a-maleimidoacetoxy)-succinimide ester (AMAS), succinimidyl-6-(P-maleimidopropionamido)hexanoate (SMPH), N-succinimidyl-4-(p- maleimidophenyl)-butyrate (SMPB), N-(p-maleimidophenyl)isocyanate (PMPI), 6- maleimidocaproyl (MC), maleimidopropanoyl (MP), p-aminobenzyloxycarbonyl (PAB), N- succinimidyl-4-(2-pyridylthio)pentanoate (SPP), N-succinimidyl (4- iodoacetyl)aminobenzoate (SIAB), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro- 2,4-dinitrobenzene). Carbon- 14-labeled l-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to the antibody (see, e.g., PCT publication number WO94 / 11026).

[0148] Exemplary methods for preparing ADCs are described in US Patent No. 7,498,298, which is expressly incorporated herein by reference.III. Therapeutic Applications

[0149] In another embodiment, the CEACAM5 binding agents described herein may be used for treating cancer, such as a solid tumor or tumor characterized by overexpression of CEACAM5. In one embodiment, a method for treating cancer comprises administering to a subject in need thereof an effective amount of a CEACAM5 binding agent described herein, optionally in combination with one or more additional exogenous anti-cancer gene products. In another embodiment, a method for treating cancer comprises administering to a subject in need thereof an effective amount of an antibody-drug conjugate (ADC) comprising a cytotoxic drug, a linker, and a CEACAM5 binding agent described herein. In another embodiment, a method for treating cancer comprises administering to a subject in need thereof an effective amount of one or more vectors expressing a CEACAM5 binding agent described herein. In a further embodiment, a method for treating cancer comprises administering to a subject in need thereof an effective amount of a population of genetically engineered T cells or NK cells expressing the CEACAM5 binding agents described herein (adoptive cell therapy). In any of these instances, one or more of the CEACAM5 binding agents or genetically engineered T cell or NK cell populations may be mixed with a pharmaceutically acceptable carrier to form apharmaceutical composition prior to administration, which is also within the scope of the present disclosure.(A ) Pharmaceutical Compositions

[0150] The therapeutically active agents described above can be mixed with a pharmaceutically acceptable carrier (excipient) to form a pharmaceutical composition for use in treating cancer. “Acceptable” means that the carrier must be compatible with the active ingredient of the composition (and preferably, capable of stabilizing the active ingredient) and not deleterious to the subject to be treated. Pharmaceutically acceptable excipients (carriers) including buffers, which are well known in the art. See, e.g., Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover.

[0151] The pharmaceutical compositions to be used in the present methods can comprise pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions. (Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations used, and may comprise buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrans; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

[0152] In some examples, the pharmaceutical composition described herein comprises liposomes containing the antibodies (or the encoding nucleic acids) which can be prepared by methods known in the art, such as described in Epstein, et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang, et al., Proc. Natl. Acad. Sci. USA 77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosedin U.S. Pat. No. 5,013,556. Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.

[0153] The antibodies, or the encoding nucleic acid(s), may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxy methylcellulose or gelatin-microcapsules and poly- (methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are known in the art, see, e.g., Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing (2000).

[0154] In other examples, the pharmaceutical composition described herein can be formulated in sustained-release format. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl- methacrylate), or poly(vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and 7 ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.

[0155] The pharmaceutical compositions to be used for in vivo administration must be sterile. This is readily accomplished by, for example, filtration through sterile filtration membranes. Therapeutic antibody compositions are generally placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

[0156] The pharmaceutical compositions described herein can be in unit dosage forms such as tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories, for oral, parenteral, or rectal administration, or administration by inhalation or insufflation.

[0157] For preparing solid compositions such as tablets, the principal active ingredient can be mixed with a pharmaceutical carrier, e.g., conventional tableting ingredients such as com starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalciumphosphate or gums, and other pharmaceutical diluents, e.g., water, to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention, or a non-toxic pharmaceutically acceptable salt thereof. When referring to these pre-formulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills, and capsules. This solid pre-formulation composition is then subdivided into unit dosage forms of the type described above containing from 0.1 to about 500 mg of the active ingredient of the present invention. The tablets or pills of the novel composition can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer that serves to resist disintegration in the stomach and permits the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.

[0158] Suitable surface-active agents include, in particular, non-ionic agents, such as polyoxyethylenesorbitans (e.g., Tween™ 20, 40, 60, 80 or 85) and other sorbitans (e.g., Span™ 20, 40, 60, 80 or 85). Compositions with a surface-active agent will conveniently comprise between 0.05 and 5% surface-active agent, and can be between 0.1 and 2.5%. It will be appreciated that other ingredients may be added, for example mannitol or other pharmaceutically acceptable vehicles, if necessary.

[0159] Suitable emulsions may be prepared using commercially available fat emulsions, such as Intralipid™, Liposyn™, Infonutrol™, Lipofundin™ and Lipiphysan™. The active ingredient may be either dissolved in a pre-mixed emulsion composition or alternatively it may be dissolved in an oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil or almond oil) and an emulsion formed upon mixing with a phospholipid (e.g., egg phospholipids, soybean phospholipids or soybean lecithin) and water. It will be appreciated that other ingredients may be added, for example glycerol or glucose, to adjust the tonicity of the emulsion. Suitable emulsions will typically contain up to 20% oil, for example,between 5 and 20%. The fat emulsion can comprise fat droplets between 0.1 and 1.0 m, particularly 0.1 and 0.5 pm, and have a pH in the range of 5.5 to 8.0.

[0160] The emulsion compositions can include those prepared by mixing an antibody with Intralipid™ or the components thereof (soybean oil, egg phospholipids, glycerol, and water).

[0161] Pharmaceutical compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous, or organic solvents, or mixtures thereof, and powders. The liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as set out above. In another embodiment, the compositions are administered by the oral or nasal respiratory route for local or systemic effect.

[0162] Compositions in preferably sterile pharmaceutically acceptable solvents may be nebulized by use of gases. Nebulized solutions may be breathed directly from the nebulizing device or the nebulizing device may be attached to a face mask, tent or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions may be administered, preferably orally or nasally, from devices which deliver the formulation in an appropriate manner.(B) Therapeutic Methods

[0163] To practice the therapeutic methods described herein, an effective amount of a pharmaceutical composition comprising a CEACAM5 binding agent, CEACAM5 drug conjugate, CEACAM5 expressing polynucleotide, or population genetically engineered T cells or NK cells expressing a CEACAM5 binding agent is administered to a subject in need of cancer treatment via a suitable route, such as intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra- articular, intrasynovial, intrathecal, oral, inhalation or topical routes. Commercially available nebulizers for liquid formulations, including jet nebulizers and ultrasonic nebulizers are useful for administration. Liquid formulations can be directly nebulized and lyophilized powder can be nebulized after reconstitution. Alternatively, the antibodies as described herein can be aerosolized using a fluorocarbon formulation and a metered dose inhaler, or inhaled as a lyophilized and milled powder.

[0164] A pharmaceutical composition comprising genetically engineered T cells or NK cells expressing the CEACAM5 binding agents is generally administered intravenously. In another embodiment, the administered T cells or NK cells are autologous to the subject, e.g.,obtained from the subject in need of the treatment, modified to express the CEACAM5 binding agent and optionally one or more additional exogenous gene products. The resultant modified cells activated and / or expanded ex vivo and then administered to the same subject. Administration of autologous cells to a subject may result in reduced rejection of the immune cells as compared to administration of non-autologous cells.

[0165] The subject to be treated may be a mammal (e.g., human, mouse, pig, cow, rat, dog, guinea pig, rabbit, hamster, cat, goat, sheep, or monkey) suffering from cancer, particularly a human patient with a cancer characterized by overexpression of CEACAM5.

[0166] The term “an effective amount” as used herein refers to the amount of each active agent required to confer therapeutic effect on the subject, either alone or in combination with one or more active agents. Effective amounts may vary, as recognized by those skilled in the art, depending on the particular condition being treated, the severity of the condition, toxicity consideration, previous trial results, individual patient parameters including age, physical condition, size, gender and weight, the duration of treatment, route of administration, excipient usage, co-usage (if any) with other active agents and like factors within the knowledge and expertise of the health practitioner. The quantity to be administered depends on the subject to be treated, including, for example, the capacity of the individual's immune system to produce a cell-mediated immune response. Effective amounts of the therapeutically active agents required to be administered depends on the judgment of the practitioner. However, suitable dosage ranges are readily determinable by one skilled in the art.

[0167] The term “treating” as used herein refers to the application or administration of a CEACAM5 binding agent, CEACAM5 ADC, CEACAM5 expressing polynucleotide, or cell population of genetically engineered immune cells expressing the CEACAM5 binding agent to a subject with cancer with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, affect progression of the cancer and its symptoms.

[0168] A subject having a target cancer can be identified by routine medical examination, e.g., laboratory tests, organ functional tests, CT scans, or ultrasounds. In another embodiment, the subject to be treated by the method described herein may be a human cancer patient who has undergone or is subjecting to an anti-cancer therapy, for example, chemotherapy, radiotherapy, immunotherapy, or surgery.

[0169] In another embodiment, the cancer for treatment is a solid tumor. In another embodiment, the solid tumor is a carcinoma. In certain embodiments, the carcinoma is an adenocarcinoma or squamous cell carcinoma thereof. Exemplary carcinomas include smallcell lung carcinoma, non-small cell lung carcinoma, squamous cell lung carcinoma, large cell lung carcinoma, pancreatic carcinoma, pancreatic ductal carcinoma, prostate carcinoma, esophageal carcinoma, breast carcinoma, ovarian carcinoma, prostate carcinoma, colorectal carcinoma, bladder carcinoma, cervical carcinoma, hepatocellular carcinoma, renal hepatocellular carcinoma, gastric carcinoma, papillary carcinoma, adrenocortical carcinoma, pituitary carcinoma, a head and neck carcinoma, an adenocarcinomas thereof, a squamous cell carcinomas thereof, and a metastatic cancer thereof. In another embodiment, the solid cancer is a glioblastoma or mesothelioma.

[0170] In another embodiment, the solid tumor expresses one or more antigens selected from the group consisting of: ICAM-1, EpCAM, CEACM5, EGFRvIII, mesothelin, CS-1, GD2, Tn Ag, PSMA, TAG72, CD44v6, CEA, KIT, IL-13Ra2, GD3, CD171, IL-1 IRa, PSCA, VEGFR2, Lewis Y, CD24, PDGFR-beta, SSEA-4, folate receptor alpha, ERBB2, Her2 / neu, MUC1, EGFR, NCAM, Ephrin B2, CAIX, LMP2, sLe, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, FAP, Legumam, HPV E6 or E7, CLDN6, TSHR, GPRC5D, ALK, Polysialic acid, PLAC1, globoH, NY-BR-1 , UPK2, HAVCR1, ADRB3, PANX3, GPR20, Ly6k, OR51E2, TARP, and GFRa4. In another embodiment, these antigens may serve as target antigens for the bispecific CEACAM5 binding agents or as secondary target antigen binding domain in conjunction with the CEACAM5 binding agents disclosed herein.

[0171] Empirical considerations, such as the half-life, generally will contribute to the determination of the dosage. For example, humanized CEACAM5 binding agents or CEACAM5 ADCs thereof which are compatible with the human immune system may be used to prolong the half-life of the CEACAM5 binding agent or CEACAM5 ADC and to prevent the CEACAM5 binding agent or CEACAM5 ADC from being attacked by the host's immune system. In addition, CEACAM5 binding agents or CEACAM5 ADCs containing FcRn binding domains (e.g., Fc domains) can additionally serve to provide increased half-life in vivo. Frequency of administration may be determined and adjusted over the course of therapy, and is generally, but not necessarily, based on treatment and / or suppression and / or amelioration and / or delay of a target disease / disorder. Alternatively, sustained continuous release formulations of the CEACAM5 binding agent or CEACAM5 ADC may be appropriate. Various formulations and devices for achieving sustained release are known in the art.

[0172] In one example, dosages for an CEACAM5 binding agent or CEACAM5 ADC as described herein may be determined empirically in individuals who have been given one or more administration(s) of the CEACAM5 binding agent or CEACAM5 ADC.Individuals are given incremental dosages of the agonist. To assess efficacy of the agonist, an indicator of the disease / disorder can be followed.

[0173] For the purpose of the present disclosure, a typical daily dosage might range from about any of 0.1 pg / kg to 3 pg / kg to 30 pg / kg to 300 pg / kg to 3 mg / kg, to 30 mg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of symptoms occurs or until sufficient therapeutic levels are achieved to alleviate a target disease or disorder, or a symptom thereof. An exemplary dosing regimen comprises administering an initial dose of about 2 mg / kg, followed by a weekly maintenance dose of about 1 mg / kg of the CEACAM5 binding agent or CEACAM5 ADC, or followed by a maintenance dose of about 1 mg / kg every other week. However, other dosage regimens may be useful, depending on the pattern of pharmacokinetic decay that the practitioner wishes to achieve. For example, dosing from one-four times a week is contemplated. In another embodiment, dosing ranging from about 3 pg / mg to about 2 mg / kg (such as about 3 pg / mg, about 10 pg / mg, about 30 pg / mg, about 100 pg / mg, about 300 pg / mg, about 1 mg / kg, and about 2 mg / kg) may be used. In another embodiment, dosing frequency is once every week, every 2 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, or every 10 weeks; or once every month, every 2 months, or every 3 months, or longer. The progress of this therapy is easily monitored by conventional techniques and assays. The dosing regimen (including the CEACAM5 binding agent or CEACAM5 ADC) can vary over time.

[0174] In another embodiment, for an adult patient of normal weight, doses ranging from about 0.3 to 5.00 mg / kg may be administered. In some examples, the dosage of the CEACAM5 binding agent or anti-CEACAM5 ADC comprising such as described herein can be 10 mg / kg. The particular dosage regimen, i.e., dose, timing, and repetition, will depend on the particular individual and that individual's medical history, as well as the properties of the individual agents (such as the half-life of the agent, and other considerations well known in the art).

[0175] An effective amount of the genetically engineered T cells or NK cells expressing the CEACAM5 binding agents described herein (optionally in combination with one or more exogenous anti-cancer gene products) may be administered to a subject in need of the treatment via a suitable route, such as intravenous administration. In another embodiment, the genetically engineered T cells, or NK cells for use in the treatment disclosed herein may beautologous to the subject, i.e., the immune cells may be obtained from the subject in need of the treatment, and then administered to the same subject. In certain embodiments, prior to re- introduction into the subject, the autologous T or NK cells are activated and / or expanded ex vivo. Administration of autologous cells to a subject may result in reduced rejection of the host cells as compared to administration of non-autologous cells.

[0176] Alternatively, the immune cells can be allogeneic cells, e.g., the cells are obtained from a first subject, modified as described herein and administered to a second subject that is different from the first subject but of the same species. For example, allogeneic immune cells may be derived from a human donor and administered to a human recipient who is different from the donor. In one embodiment, the T lymphocytes are allogeneic T lymphocytes, optionally in which the expression of the endogenous T cell receptor has been inhibited or eliminated. In certain embodiments, prior to introduction into the subject, the allogeneic T lymphocytes are activated and / or expanded ex vivo. T lymphocytes can be activated by any method known in the art, e.g., in the presence of anti-CD3 / CD28, IL-2, and / or phytohemoagglutinin.

[0177] NK cells can be activated by any method known in the art, e.g., in the presence of one or more agents selected from the group consisting of CD 137 ligand protein, CD137 antibody, IL-15 protein, IL-15 receptor antibody, IL-2 protein, IL-12 protein, IL-21 protein, and K562 cell line. See, e.g., U.S. Patents Nos. 7,435,596 and 8,026,097 for the description of useful methods for expanding NK cells. For example, NK cells used in the methods of the disclosure may be preferentially expanded by exposure to cells that lack or poorly express major histocompatibility complex I and / or II molecules and which have been genetically modified to express membrane bound IL- 15 and 4- IBB ligand (CDI37L). Such cell lines include, but are not necessarily limited to, K562 [ATCC, CCL 243; Lozzio et al., Blood 45(3): 321-334 (1975); Klein et al., Int. J. Cancer 18: 421-431 (1976)], and the Wilms tumor cell line HFWT (Fehniger et al., Int Rev Immunol 20(3-4):503-534 (2001); Harada H, et al., Exp Hematol 32(7):614-621 (2004)), the uterine endometrium tumor cell line HHUA, the melanoma cell line HMV-II, the hepatoblastoma cell line HuH-6, the lung small cell carcinoma cell lines Lu- 130 and Lu-134-A, the neuroblastoma cell lines NB 19 and N 1369, the embryonal carcinoma cell line from testis NEC 14, the cervix carcinoma cell line TCO-2, and the bone marrow-metastasized neuroblastoma cell line TNB 1 [Harada, et al., Jpn. J. Cancer Res 93: 313-319 (2002)]. Preferably the cell line used lacks or poorly expresses both MHC I and II molecules, such as the K562 and HFWT cell lines. A solid support may be used instead of acell line. Such a support should preferably have attached on its surface at least one molecule capable of binding to NK cells and inducing a primary activation event and / or a proliferative response or capable of binding a molecule having such an affect thereby acting as a scaffold. The support may have attached to its surface the CD 137 ligand protein, a CD 137 antibody, the IL-15 protein, or an IL-15 receptor antibody. Preferably, the support will have IL-15 receptor antibody and CD 137 antibody bound on its surface.

[0178] Patients can be treated by infusing therapeutically effective doses of the genetically engineered T or NK cells in ranges of about 105to 109or 1 x 106to about 1 x 108cells to a patient. In some examples, a human patient may receive multiple doses of the immune cells. For example, the patient may receive two doses of the immune cells on two consecutive days. In some instances, the first dose is the same as the second dose. In other instances, the first dose is lower than the second dose, or vice versa.

[0179] In any of the treatment methods involving the use of the modified immune cells disclosed herein, the subject may be administered IL-2 concurrently with the cell therapy. More specifically, an effective amount of IL-2 may be given to the subject via a suitable route before, during, or after the cell therapy. In another embodiment, IL-2 is given to the subject after administration of the immune cells. In another embodiment, prior to the cell therapy, the subject receives a lymphodepleting treatment to condition the subject for the cell therapy.

[0180] In another embodiment, one or more nucleic acids capable of expressing the CEACAM5 binding agents may be administered to a patient with cancer. Therapeutic compositions containing a polynucleotide (e.g., those encoding the antibodies described herein) are administered in a range of about 100 ng to about 200 mg of DNA for local administration in a gene therapy protocol. In another embodiment, concentration ranges of about 500 ng to about 50 mg, about 1 pg to about 2 mg, about 5 pg to about 500 pg, and about 20 pg to about 100 pg of DNA or more can also be used during a gene therapy protocol.

[0181] The therapeutic polynucleotides and polypeptides described herein can be delivered using gene delivery vehicles. The gene delivery vehicle can be of viral or non- viral origin (see generally, Jolly, Cancer Gene Therapy (1994) 1:51; Kimura, Human Gene Therapy (1994) 5:845; Connelly, Human Gene Therapy (1995) 1:185; and Kaplitt, Nature Genetics (1994) 6:148). Expression of such coding sequences can be induced using endogenous mammalian or heterologous promoters and / or enhancers. Expression of the coding sequence can be either constitutive or regulated. Recombinant viruses for delivery of a desired polynucleotide and expression in a targeted cancer cell are well known in the art. Exemplaryviral-based vehicles for in vivo delivery include, but are not limited to, recombinant retroviruses, lentiviruses, adeno-associated viruses (AAVs), adenoviruses, and alphaviruses. In another embodiment, non-viral delivery vehicles, including naked DNA, nanoparticles, and liposomes. Compositions and methods for in vivo delivery polynucleotides and polypeptides are well known in the art.

[0182] In another embodiment, the CEACAM5 binding agents, anti-CEACAM5 CEACAM5 ADCs, genetically engineered T cells or NK cells expressing the CEACAM5 binding agents, or polynucleotides may be administered in conjunction with other types of therapy for cancer, such as chemotherapy, surgery, radiation, gene therapy, and so forth. Such therapies can be administered simultaneously or sequentially (in any order) with the immunotherapy according to the present disclosure. When co-administered with an additional therapeutic agent, suitable therapeutically effective dosages for each agent may be lowered due to the additive or synergistic effects.(C) Co-Administration of Tyrosine Kinase Inhibitors.

[0183] Treatment with T or NK cell engager agents, as exemplified by the BiTEs, NKCEs, and genetically engineered T and NK cells described herein, is sometimes associated with safety liabilities due to on-target on-tumor, on-target off-tumor cytotoxic activity and cytokine release. One of the most common adverse effects reported for T cell engaging agents is Cytokine Release Syndrome (CRS).

[0184] Therefore, in another embodiment, tyrosine kinase inhibitors may be used to mitigate CRS due to such T or NK cell engaging therapies. For example, it is known that transient inhibition or modulation of TCR signaling and / or CAR signaling in human T cells can prevent or reverse T cell exhaustion and restore T cell function to CAR-T cells undergoing functional exhaustion. In particular, in vivo treatment of the CAR-T cells described herein with certain tyrosine kinase inhibitors inhibiting T cell receptor signaling (e.g., a Lek tyrosine kinase inhibitor (e.g., dasatinib)) can suppress exhaustion marker expression, augment memory formation, decrease expression of PD1 on CAR-T cells, and facilitate cell survival / proliferation as described in US 2020 / 101108 Al and US 2021169880 Al, the disclosures of which are expressly incorporated by reference herein. Similar findings have been obtained using Src, mTOR, JAK inhibitors or small molecules having a thiazole, imidazolepyridiazine orpiperazinyl-methyl-aniline structure See e.g., US 2021 / 0393628 Al, WO 2022 / 0168418, WO 2022 / 223651, incorporated by reference herein.

[0185] Thus, in another embodiment, a patient receiving a CEACAM5 binding agent or cell therapy in accordance with the present disclosure may be additionally administered a tyrosine kinase inhibitor, such as a Src, mTOR or JAK inhibitor (e.g., or a small molecule having a thiazole, imidazolepyridiazine or piperazinyl-methyl-aniline structure to mitigate T cell exhaustion, augment memory T cell formation, and / or maintain and facilitate cell survival and proliferation. In another embodiment, these active agents may be used to improve ex vivo cell expansion and collection of genetically engineered cells that are resistant and / or less prone to T or NK cell exhaustion. Thus, in another aspect, the present disclosure further provides cell compositions comprising genetically engineered cells that are expanded in the presence of particular compounds described herein.

[0186] Exemplary tyrosine kinase inhibitors for administration include Src- and / or Bcr-Abl inhibitors include dasatinib, ponatinib, saracatinib, bosutinib, and nilotinib. Exemplary JAK inhibitors include ruxolitinib, baricitinib, momelotinib, upadacitinib, filgotinib, abrocitinib, itacitinib, solcitinib, oclacitinib, fedratinib, gandotinib, lestaurtinib and pacritinib. Exemplary mTOR inhibitors include sirolimus, temsirolimus, everolimus and ridaforolimus. Exemplary small molecules having a thiazole, imidazolepyridiazine or piperazinyl-methyl-aniline structure are disclosed in US 2021 / 0393628 Al, the disclosures of which are expressly incorporated by reference herein.

[0187] Such methods are not limited to particular manner of administration. In another embodiment, multiple cycles of treatment are administered to the subject. In another embodiment, the pharmaceutical composition is administered intermittently. In another embodiment, the pharmaceutical composition is administered for a period of time sufficient to restore at least partial T cell function then discontinued. In another embodiment, the pharmaceutical composition is administered orally.

[0188] In another embodiment, the pharmaceutical compositions are administered iteratively for purposes of facilitating periods of inactivation of e.g., genetically engineered T or NK cells (e.g., during pharmaceutical composition administration) and periods of their activation (e.g., during absence of pharmaceutical composition administration; following clearance of the pharmaceutical composition).

[0189] In another embodiment, patients undergoing treatment are subjected to intermittent exposure to dasatinib (or other active agents above) to reduce exhaustion, andaugment the engraftment, proliferation and persistence of the genetically engineered T or NK cells in vivo., as well as antitumor function of these cells.

[0190] The terms “intermittent administration” or “administered intermittently” in connection with the tyrosine kinase inhibitors described herein refer to the use of these tyrosine kinase inhibitors in an administration regime that causes intermittent changes between a state wherein the patient has tyrosine kinase inhibitor serum levels within the therapeutic window and a state wherein the patient has tyrosine kinase inhibitor serum levels below the therapeutic window. A therapeutic window of a given tyrosine kinase inhibitor can be determined by any methods known in the art.

[0191] Alternatively, the terms “intermittent administration” and “administered intermittently” in connection with a tyrosine kinase inhibitor as used herein refer to the use of a tyrosine kinase inhibitor in an administration regime causing: (1) intermittent changes between a state where the patient has tyrosine kinase inhibitor serum levels causing complete inhibition of the tyrosine kinase and a state where the patient has tyrosine kinase inhibitor serum levels causing partial inhibition of the tyrosine kinase; (2) intermittent changes between a state where the patient has tyrosine kinase inhibitor serum levels causing complete inhibition of the tyrosine kinase and a state where the patient has tyrosine kinase inhibitor serum levels causing no inhibition of the tyrosine kinase; or (3) intermittent changes between a state where the patient has tyrosine kinase inhibitor serum levels causing partial inhibition of the tyrosine kinase and a state where the patient has tyrosine kinase inhibitor serum levels causing no inhibition of the tyrosine kinase.

[0192] Such inhibition can be measured by any methods known in the art, e.g., by measuring the activity of the tyrosine kinase itself using appropriate enzyme assays, or by measuring cellular functions downstream of the kinase. In another embodiment, a partial inhibition refers to an inhibition of at least 25% to 75% compared to a situation in the absence of the inhibitor. As used herein, “no inhibition” refers to an inhibition of less than 25%, or less than 10% compared to a situation in the absence of the inhibitor.

[0193] In the case of genetically engineered T or NK cells, inhibition of less than 25% or 10% can be an inhibition of the cytotoxic lysis, cytokine secretion, and / or proliferation of the T cells. Further, the inhibition of at least 25%, but no more than 75%, can preferably be an inhibition of the cytotoxic lysis, cytokine secretion, and proliferation of the T or NK cells.

[0194] In another embodiment, intermittent administration of dasatinib may cause intermittent changes between a state wherein the serum levels of dasatinib are above 50 nMand a state wherein the serum levels of dasatinib are at or below 50 nM. Intermittent administration may be achieved by using an administration interval longer than the terminal phase half-life of the tyrosine kinase inhibitor, longer than 2 times the terminal phase half-life of the tyrosine kinase inhibitor, or longer than 3 times, 4 times, or 5 times the terminal phase half-life of the tyrosine kinase inhibitor. For example, intermittent administration of dasatinib may be achieved using an administration interval of at least 6 hours for dasatinib or at least 12 hours for dasatinib. It will be understood by a person skilled in the art that for each administration regime, appropriate dosages of the respective tyrosine kinase inhibitors can be selected based on pharmacokinetic and pharmacodynamic experiments.

[0195] The terms “continuous administration” or “administered continuously” in connection with a tyrosine kinase inhibitor as used herein refer to the use of said tyrosine kinase inhibitor in an administration regime that causes a complete inhibition of the tyrosine kinase in a continuous manner. According to the invention, a complete inhibition refers to an inhibition of at least 75%, compared to a situation in the absence of the inhibitor. Such inhibition can be measured by any methods known in the art, e.g., by measuring the activity of the tyrosine kinase itself using appropriate enzyme assays, or by measuring cellular functions downstream of said kinase.

[0196] In the case of the genetically engineered T or NK cells, inhibition of at least 75% can be an inhibition of the cytotoxic lysis, cytokine secretion, and proliferation of T or NK cells. Alternatively, the terms “continuous administration” and “administered continuously” in connection with a tyrosine kinase inhibitor described herein refer to use of the tyrosine kinase inhibitor in an administration regime that results in serum levels of the tyrosine kinase which are continuously within the therapeutic window. In another embodiment, continuous administration of dasatinib encompasses any administration wherein the serum levels of dasatinib are constantly maintained at or above 50 nM. In one embodiment, dasatinib is administered continuously, where the administration comprises oral administration of 50- 200 mg dasatinib every 6-8 hours or 140 mg every 6 hours.

[0197] In another embodiment, the threshold serum level is within the range of 0.1 nM-1 pM, 1 nM-500 nM, 5 nM-100 nM, 10 nM-75 nM, or 25 nM-50 nM.(D) Monitoring Genetically Engineered T or NK Cell Distribution in a Patient

[0198] In another aspect, the present disclosure provides a method for treating cancer and monitoring genetically engineered T or NK cell distribution in a patient undergoingcell therapy with these cells as disclosed herein. The method involves co-expressing human somatostatin receptor 2 (SSTR2) in the genetically engineered T or NK cells as a cell surface marker for monitoring distribution of these cells following their introduction in a patient.

[0199] SSTR2 can be used in conjunction with FDA-approved positron emission tomography (PET) radiotracers currently used in clinics to probe for overexpressed SSTR2 in neuroendocrine tumors, specifically68Gallium conjugates of DOTATOC and DOTATATE. Single-photon emission computed tomography (SPECT)-based imaging is also available usingi nIn-DTPAOC (Octreoscan) or177Lutetium. SSTR2 displays restricted basal expression in tissues and all major organs except in the kidneys and cerebrum making it ideal for detection of adoptively transferred T or NK cells targeting a multitude of CEACAM5 overexpressing solid tumors.

[0200] It has previously been shown that SSTR2 facilitates rapid radiotracer uptake and this combined with swift renal clearance of unbound DOTATOC means that high quality, clinical-grade images can be obtained at one hour post DOTATOC injection. DOTATOC also has a short half-life of 68 min which, combined with its rapid clearance, delivers a low radiation dose to the patient. The fact that SSTR2 is of human origin also limits its immunogenicity which has plagued experiments using non-human genetic reporters.

[0201] SSTR2 compositions and methods for using SSTR2 as a reporter for T or NK cell monitoring and use in cancer are disclosed in U.S. Patent No. 10,577,408, which is expressly incorporated by reference herein.

[0202] In one embodiment, the method comprises: (a) incubating a population of genetically engineered T or NK cells described herein with a radioactive label that binds to SSTR2; (b) intravenously infusing the labeled cells into a patient in an amount of 104-108cells / kg patient, and (c) detecting the labeled T or NK cell distribution by positron emission tomography / computed tomography (PET / CT) imaging, wherein the labeled cells are infiltrated into cancer cells to kill the cancer cells. In this method, SSTR2 is pre-labeled in vitro. In another embodiment, the labeled cells are administered in an amount of 106-108or 106- 107cells / kg of the patient.

[0203] In another embodiment, the method comprises: (a) intravenously infusing a population of genetically engineered T or NK cells described herein into a patient; (b) injecting into the patient a radioactive label that binds to SSTR2 at least one hour prior to PET / CT imaging, and (c) detecting the labeled cell distribution by PET / CT imaging, wherein the labeled cells are infiltrated into cancer cells to kill the cancer cells. In this method, SSTR2 is labeledpost-infusion in vivo. In a further embodiment, the genetically engineered T or NK cells have been transduced to express at least 100,000 molecules of SSTR2 per T cell.

[0204] In another embodiment, the label is radioactively labeled DOTATOC or radioactively labeled DOTATATE, such as68Gallium-DOTATOC or68Gallium-DOTATATE. The methods for monitoring distribution of radiolabeled T or NK cells may be used in connection with treating any of the cancers described herein, including but not limited to e.g., lung cancer, gastric cancer, thyroid cancer, pancreatic cancer, or breast cancer.(E) Combination Therapies

[0205] The CEACAM5 binding agents, anti-CEACAM5 ADCs, genetically engineered T cells, NK cells, or polynucleotides expressing the CEACAM5 binding agents described herein may be administered conjunction with other types of therapy or active agents for cancer, such as chemotherapy, surgery, radiation, gene therapy, and so forth. Such therapies can be administered simultaneously or sequentially (in any order) with the immunotherapy described herein. When co-administered with an additional therapeutic agent, suitable therapeutically effective dosages for each agent may be lowered due to additive or synergistic effects.

[0206] In some examples, the subject is treated with an anti-cancer therapy to reduce tumor burden prior to the therapy disclosed herein. For example, the subject (e.g., a human cancer patient) may be treated with chemotherapy (e.g., comprising a single chemotherapeutic agent or a combination of two or more chemotherapeutic agents) at a dose that substantially reduces tumor burden. In some instances, the chemotherapy may reduce the total white blood cell count in the subject to lower than 108 / L, e.g., lower than 107 / L. Tumor burden of a patient after the initial anti-cancer therapy, and / or after the therapy disclosed herein may be monitored via routine methods. If a patient showed a high growth rate of cancer cells after the initial anti-cancer therapy and / or after the CEACAM5-directed therapy disclosed herein, the patient may be subjected to a new round of chemotherapy to reduce tumor burden.

[0207] Non-limiting examples of other anti-cancer therapeutic agents for use in combination with the CEACAM5 binding agents, anti-CEACAM5 ADCs, or genetically engineered T cells or NK cells expressing the CEACAM5 binding agents described herein include, but are not limited to, immune checkpoint inhibitors (e.g., PDL1, PD1, and CTLA4 inhibitors), anti-angiogenic agents (e.g., TNP-470, platelet factor 4, thrombospondin- 1, tissue inhibitors of metalloproteases, prolactin, angiostatin, endostatin, bFGF soluble receptor,transforming growth factor beta, interferon alpha, interferon gamma, soluble KDR and FLT-1 receptors, and placental proliferin-related protein); VEGF antagonists (e.g., anti-VEGF antibodies, VEGF variants, soluble VEGF receptor fragments); chemotherapeutic compounds. In another embodiment, the anti-cancer therapeutic agents include pembrolizumab (Keytruda™), ipilimumab (Yervoy™), nivolumab (Opdivo™), or atezolizumab (Tecentriq™).

[0208] Exemplary chemotherapeutic compounds include pyrimidine analogs (e.g., 5-fluorouracil, floxuridine, capecitabine, gemcitabine and cytarabine); purine analogs (e.g., fludarabine); folate antagonists (e.g., mercaptopurine and thioguanine); antiproliferative or antimitotic agents, for example, vinca alkaloids; microtubule disruptors such as taxane (e.g., paclitaxel, docetaxel), vincristin, vinblastin, nocodazole, epothilones and navelbine, and epidipodophyllotoxins; and DNA damaging agents (e.g., actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethyhnelamineoxaliplatin, iphosphamide, melphalan, merchlorehtamine, mitomycin, mitoxantrone, nitrosourea, plicamycin, procarbazine, taxol, taxotere, teniposide, triethylenethiophosphoramide and etoposide).

[0209] In another embodiment, radiation or radiation and chemotherapy is used in combination with the cell populations comprising modified immune cells described herein. Additional useful agents and therapies can be found in Physician’s Desk Reference, 59thedition, (2005), Thomson P D R, Montvale N.J.; Gennaro et al., Eds. Remington’s The Science and Practice of Pharmacy 20thedition, (2000), Lippincott Williams and Wilkins, Baltimore Md.; Braunwald et al., Eds. Harrison’s Principles of Internal Medicine, 15thedition, (2001), McGraw Hill, NY; Berkow et al., Eds. The Merck Manual of Diagnosis and Therapy, (1992), Merck Research Laboratories, Rahway N J.IV. Kits for Therapeutic Uses

[0210] The present disclosure also provides kits for any of the therapeutic uses described herein. In another embodiment, the kit comprises one or more CEACAM5 binding agents, polynucleotides for expressing CEACAM5 binding agents, CEACAM5 ADCs, orgenetically engineered T cells or NK cells described herein, as well as kits for use in making such therapeutically active agents described herein.

[0211] A kit for therapeutic use as described herein may include one or more containers comprising any of the therapeutically active agents above, which may be in combination with one or more excipients for forming a pharmaceutical composition. In another embodiment, the kit can additionally comprise instructions for use of the above therapeutic agents in any of the methods described herein. The included instructions may comprise a description of administration of a pharmaceutical composition comprising the therapeutically active agent to a subject to achieve the intended result in a subject. The kit may further comprise a description of selecting a subject suitable for treatment based on identifying whether the subject is in need of the treatment. In another embodiment, the instructions include a description of administering the pharmaceutical composition comprising the therapeutically active agent to a subject who is in need of the treatment.

[0212] The instructions relating to the use of a pharmaceutical composition comprising the therapeutically active agent as described herein generally include information as to dosage, dosing schedule, and route of administration for the intended treatment. The containers may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses. Instructions supplied in the kits of the disclosure are typically written instructions on a label or package insert. The label or package insert indicates that the pharmaceutical compositions are used for treating, delaying the onset, and / or alleviating a disease or disorder in a subject.

[0213] The kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, and the like. Also contemplated are packages for use in combination with a specific device, such as infusion device. A kit may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The container may also have a sterile access port.

[0214] Kits optionally may provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container. In some embodiment, the disclosure provides articles of manufacture comprising contents of the kits described above.

[0215] Also provided here are kits for use in making the genetically engineered T or NK cells as described herein. Such a kit may include one or more containers each containing reagents for use in introducing the CEACAM5 binding agents and optionally other exogenousproducts into T or NK cells, as well as cell culture components for growth and expansion of the cells. Such a kit may further include instructions for making desired modifications to nucleic acids or host T or NK cells using components supplied in the kit.V. General Techniques

[0216] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait, ed. 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1989) Academic Press; Animal Cell Culture (R. I. Freshney, ed. 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds. 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.): Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds. 1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds. 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practice approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds. Harwood Academic Publishers, 1995); DNA Cloning: A practical Approach, Volumes I and II (D.N. Glover ed. 1985); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds. (1985; Transcription and Translation (B.D. Hames & S.J. Higgins, eds. (1984; Animal Cell Culture (R.I. Freshney, ed. (1986; Immobilized Cells and Enzymes (IRL Press, (1986); B. Perbal, A practical Guide To Molecular Cloning (1984); F.M. Ausubel et al. (eds.); Chimeric Antigen Receptor (CAR) Immunotherapy (D. W. Lee and N. N. Shah, eds., Elsevier, 2019, ISBN:9780323661812); Basics of Chimeric Antigen Receptor (CAR) Immunotherapy (M. Y. Balkhi, Academic Press,Elsevier Science, 2019, ISBN:9780128197479); Chimeric Antigen Receptor T Cells Development and Production (V. Picango-Castro, K. C. R. Malmegrim, K.Swiech, eds., Springer US, 2020, ISBN:9781071601488); Cell and Gene TherapieslC. Bollard, S. A. Abutalib, M.-A. Perales eds., Springer International, 2018; ISBN:9783319543680) and Developing Costimulatory Molecules for Immunotherapy of Diseases (M. A. Mir, Elsevier Science, 2015, ISBN:9780128026755).

[0217] The present disclosure is not limited in its application to the details of construction and the arrangements of component set forth in the description herein or illustrated in the drawings. The present disclosure is capable of other embodiments and of being practice or of being carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” or “having,” “containing,” “involving,” and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. As also used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise.

[0218] Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.EXAMPLESExample 1: Generation of Reduced Affinity CEACAM5 CAR-T Cells

[0219] Site directed mutagenesis was used to create affinity tuned anti-CEACAM5 variant single-chain variable fragments (scFvs) comprising variable heavy (Vn) regions with lower affinities than scFvs comprising parental versions of the VH and variable light (VL) chain regions. Edits to the CDR3 of the heavy chain were made using an NEB Q5 site-directed mutagenesis kit (NEB#E0554S) with individually designed primers outlined in Table 1 to create desired amino acid changes.Table 1. Primers used for making antibodies*mutant nucleotides are in small case

[0220] Lentivirus constructs were prepared to facilitate characterization of the binding properties of the affinity tuned anti-CEACAM5 scFvs in chimeric artificial receptors (CARs). CEACAM5 CARs were prepared by cloning genetic sequences encoding parental (wild-type) Affy58 and Affy69 scFvs and the affinity-tuned Affy58 and Affy69 variant scFvs into a lentiviral vector between a c-myc tag and CAR element CD8 hinge. As shown in FIG. l, the lentiviral vector construct comprises (5’ to 3’) the EFl a promoter, c-myc tag, CEACAM5 scFv, CD8 hinge, CD28 transmembrane domain, CD28 co-stimulatory domain, CD3 zeta signaling domain, porcine teschovirus-1 2A (P2A) ribosome skipping element, and a human somatostatin receptor 2 (SSTR2) reporter gene for CAR-T cell imaging.

[0221] To obtain stably transformed CAR-expressing cells, lentivirus particles were produced by transiently transfecting HEK 293T cells using the Tran5lT®-Eenti transfection reagent, (Minis Bio, MIR6604). Briefly, 8 pg of transfer gene and 12 pg of EV- MAX lentiviral packaging mix (ThermoFisher, A43237) were mixed with Opti-MEM and 50 pL of Trans-IT and incubated for 20 minutes at room temperature. The resulting solutions were added dropwise to 10 cm2cell culture dishes seeded with 6xl06HEK 293T cells in 10 mL DMEM 24 hrs. previously. Transfection media was replaced 16-24 hrs. later. Media containing lentivirus was harvested at 48 hrs. post transfection, filtered through 0.45 pm filers and concentrated by adding Lenti-X concentrator (Takara, 631231) at 1 / 3 of final volume of supernatant. The solution was then incubated a 4 °C for at least 3 hrs. (as long as overnight) and then spun at 1,500g for 1 hr at 4 °C. The supernatant was removed, and the pellet was resuspended in 400 pL of sterile PBS and stored at -80 °C.Example 2: Evaluation of Binding Properties of Affinity-Tuned CEACAM5 CARs

[0222] Binding of labeled CEACAM5 to CAR-T cells expressing the affinity tuned CEACAM5 scFvs was evaluated by transducing lentivirus anti-CEACAM5 CAR particles into human T cells to form CEACAM5 CAR-T cells incubated with AF647-labeled CEACAM5. Briefly, human T cells were transduced 24 hrs. post activation with ImmunoCult™ CD3 / CD28 T cell activator (StemCell, 10971) in 10 mF of media containing IL7 (12.5 ng / mL), IL15 (12.5 ng / mL) and 10 pL of Lenti-BOOST (Mayflower Bioscience, SBPLV10103) in G-Rex® 6M wells (Scale Ready, 80660M). Following a 48 hr. incubation at 37 °C, the culture media was brought up to 100 mF per well and incubated at 37 °C for 10 days.

[0223] Binding curves were generated by serial diluting AF647 labeled CEACAM5 protein (AlexaFluor™ 647 antibody labeling kit, ThermoFisher, A20186) and staining each sample with c-myc antibody (Miltenyi, 130-116-485) at a 1:800 dilution. Cells were stained on ice for 30 mins and then washed with PBS. After washing, residually bound protein on CAR T cells were measured on a flow cytometer (Beckman, CytoFEEX®). The resulting binding data was fit using a non-linear regression analysis on GraphPad Prism to calculate half effective dose concentration (EC50) values. The results of this analysis are shown in FIGs. 2- 3 and Tables 2-3 below.

[0224] As compared to CAR-T cells expressing the parental anti-CEACAM5 scFv binding domains (i.e., Affy58 and Affy69), CAR-T cells expressing the affinity-tuned anti- CEACAM5 scFvs exhibiting reduced affinities for CEACAM5 ranging between 849-fold and 28964-fold relative to the parental-expressing CARs (see FIGs. 2-3 and Tables 2-3).Table 2. Binding Affinities of Parental- and Variant Affy58-Based CEACAM5 CAR-T Cells for CEACAM5Table 3. Binding Affinities of Parental- and Variant Affy69-Based CEACAM5 CAR-T Cells for CEACAM5Example 3: Cytotoxicity of Reduced Affinity CEACAM5 CAR-T Cells in Cancer Cell Lines

[0225] One of the pitfalls of CAR-T cell therapies concerns T cell exhaustion associated with recognition of antigen both on normal, non-target cells as well as on cancer cells. However, when using reduced affinity CEACAM5 CAR-T cells to reduce cell toxicities and T cell exhaustion, it is important to maintain effective killing of the targeted cancer cells.

[0226] To evaluate cytotoxicity of the reduced affinity CEACAM5 CAR-T cells against human carcinoma cells, 5xl04target cells (SNU638, Hs746T, and H1993) stably transduced to express GFP and firefly luciferase were co-cultured with CEACAM5 CAR-T cells at an effector to target ratio of 5:1. Co-cultures were carried out in T cell culture medium containing 150 pg / mL D-Luciferin (Fisher Scientific, NC1276267) with no cytokine supplementation. Luminescence was measured using a plate reader (BioTek®, Synergy Neo2) with readings every 24 hrs. for 3 days. In each case, readings were normalized to the no T cell control group and percent cytotoxicity was reported.

[0227] As shown in FIGs. 4-7 and Tables 4-7 below, all of the CEACAM5 CAR-T cells of the present disclosure exhibited cytotoxicities similar to or moderately reduced when compared the high-affinity parental CEACAM5 CAR-T cells in the 3 carcinoma cell lines tested. Although the cytotoxicities were significantly reduced in the 24 hour co-cultures compared to the high-affinity parental CAR-T cells, upon further incubation for 48 and 72 hours, the cytotoxicities typically approached the cytotoxicity levels obtained with the high- affinity parental CAR-T cells (see FIGs. 6-9 and Tables 6-9 below).Table 4. In Vitro Cytotoxicity of Parental- and Variant Affy58-Based CEACAM5 CAR- T Cells Against LoVo Human Colon Adenocarcinoma CellsTable 5. In Vitro Cytotoxicity of Parental- and Variant Affy58-Based CEACAM5 CAR- T Cells Against HCC827 Human Lung Carcinoma CellsTable 6. In Vitro Cytotoxicity of Parental- and Variant Affy69-Based CEACAM5 CAR- T Cells Against LoVo Human Colon Adenocarcinoma CellsTable 7. In Vitro Cytotoxicity of Parental- and Variant Affy69-Based CEACAM5 CAR- T Cells Against HCC827 Human Lung Carcinoma CellsExample 4: Antibody Internalization Evaluation

[0228] To evaluate the ability of Affy69 and Affy69_W7A antibodies (IgG) to be internalized, 5x104 target cells (LoVo and 293T) were seeded using Gibco’s FluoroBrightTM DMEM (Thermofisher, A1896701) containing 10% Heat Inactivated Fetal Bovine Serum (Thermofisher, 10082147). The antibodies were labeled with Zenon™ pHrodo™ iFL IgG Labeling Reagents (Thermofisher, Z25611) and added to the plated cells at a final concentration of 1.5 ug / mL. Internalization was measured using an image reader (Incucyte® S3 Live-Cell Analysis Instrument) and was incubated at 37 °C, 5% CO2. Readings were taken every 2 hrs. for up to 3 days. Internalization was measured using the Incucyte® Base Analysis Software to determine Green Integrated Intensity per Well. In each case, readings were normalized to the no antibody wells containing pHrodo ™ iFL IgG Labeling Reagent and internalization was reported. Results are shown in FIG. 8.

[0229] Affy69 and Affy69_W7A show proof of principal for ADC development. Internalization for cell lines expressing CEACAM5 was shown for both designs. Affy69 and Affy69_W7A showed no internalization for 293T cells that do not express CEACAM5.List of Sequences

[0230] Exemplary amino acid sequences described herein are provided in Table 8 below:Table 8. Amino Acid- and Nucleotide Sequences Described Herein**CDR regions underlined and italicized. CDR1, CD2, and CD3 regions are underlined and italicized in the VH and VL sequences. Only HCDR3 regions are underlined and italicized in the scFv and CAR sequences. Mutated amino acids of nucleotides in the HCDR3 regions are bolded.OTHER EMBODIMENTS

[0231] All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly statedotherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

[0232] From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the claims

[0233] From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.EQUIVALENTS

[0234] While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the function and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the inventive teachings is / are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials,kits, and / or methods, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

[0235] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0236] All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

[0237] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0238] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, e.g., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, e.g., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0239] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, e.g., the inclusion of at least one, but also including more than one of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (e.g., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only oneof,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0240] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0241] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

Claims

What Is Claimed Is:

1. A CEACAM5 binding agent comprising a CEACAM5 binding domain, wherein the CEACAM5 binding domain comprises: a heavy chain variable (VH) region comprising complementary determining 1 (HC CDR1), complementary determining 2 (HC CDR2), and complementary determining 3 (HC CDR3) regions; and a light chain variable (VL) region comprising complementary determining 1 (LC CDR1), complementary determining 2 (LC CDR2), and complementary determining 3 (LC CDR3) regions, wherein:(i) the VH region comprises HC CDR1 and HC CDR2 regions comprising amino acid sequences set forth SEQ ID NOs: 1 and 2, respectively, and an HC CDR3 region comprising an amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 4; and the VL region comprises LC CDR1, LC CDR2, and LC CDR3 regions comprising amino acid sequences set forth in SEQ ID NOs: 5-7, respectively, or(ii) the VH region comprises HC CDR1 and HC CDR2 regions comprising amino acid sequences set forth in SEQ ID NOs: 34 and 35, respectively, an HC CDR3 region comprising an amino acid sequence set forth in SEQ ID NO: 36 or 37; and the VL region comprises LC CDR1, LC CDR2, and LC CDR3 regions comprising amino acid sequences set forth in SEQ ID NOs: 38-40, respectively.

2. The binding agent of claim 1, wherein the VH region comprises the amino acid sequences set forth in (i).

3. The binding agent of claim 1 or claim 2, wherein the CEACAM5 binding domain comprises VH and VL region amino acid sequences selected from the group consisting of:(a) SEQ ID NOs: 9 and 11, respectively; and(b) SEQ ID NOs: 10 and 11, respectively.

4. The binding agent of any one of claims 1-3, wherein the binding agent comprises or consists of a single chain variable fragment (scFv).

5. The binding agent of claim 4, wherein the anti-CEACAM5-scFv comprises a (GxS)n linker between the VH and VL regions in which x is an integer between 1-6, inclusive, and n is an integer between 1-10, inclusive.

6. The binding agent of claim 4, wherein the scFv comprises an amino acid sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 18.

7. The binding agent of claim 1, wherein the VH region comprises the amino acid sequences set forth in (ii).

8. The binding agent of claim 7, which comprises VH and VL region amino acid sequences selected from the group consisting of:(a) SEQ ID NOs: 42 and 44, respectively; and(b) SEQ ID NOs: 43 and 44, respectively.

9. The binding agent of claim 7 or claim 8, wherein the binding agent comprises or consists of an scFv.

10. The binding agent of claim 9, wherein the scFv comprises a (GxS)nlinker between the Vnand VL regions in which x is an integer between 1-6, inclusive, and n is an integer between 1-10, inclusive.

11. The binding agent of claim 9, wherein the scFv comprises an amino acid sequence set forth in SEQ ID NO: 50 or 51.

12. The binding agent of any one of claims 1-11, wherein the binding agent is an IgG.

13. The binding agent of claim 12, wherein the IgG is selected from the group consisting of an IgGl, an IgG2, an IgG3, an IgG4 or a variant thereof.

14. The binding agent of any one of claims 1-13, wherein the CEACAM5 binding agent is a bispecific CEACAM5 binding agent comprising the CEACAM5 binding domain and a second antigen binding domain.

15. The binding agent of claim 14, wherein the CEACAM5 binding agent is a bispecific scFv, bispecific CAR, a diabody, a tandem diabody, a DART, a minibody, an scFv- Fab, an scFv-scFab, a cross-over Fab, a Fab, an (scFv)2-Fab, a triabody, a tetrabody, or a VH- scFv,16. The binding agent of claim 14 or claim 15, wherein the second antigen binding domain is an scFv.

17. The binding agent of claim 16, wherein the second antigen binding domain binds a target antigen selected from the group consisting of ICAM-1, c-MET, EpCAM, and SSTR2.

18. The binding agent of claim 16 or claim 17, wherein the CEACAM5 binding agent is a bispecific immune cell engager.

19. The binding agent of claim 18, wherein the second antigen binding domain binds CD3, CD16, or CD40.

20. The binding agent of any one of claims 1-19, wherein the first and / or second antigen binding domain binds their target antigen with an EC50 between 5 pM and 40 p M.

21. The binding agent of any one of claims 1-20, further comprising an immunoglobulin constant region.

22. The binding agent of 21, wherein the immunoglobulin constant region comprises an IgGl CHI domain, a CH2 region, a CH3 region, or a combination thereof.

23. The binding agent of claim 22, wherein the binding agent comprises an Fc domain.

24. The binding agent of claim 23, wherein the binding agent comprises a single chain Fc domain.

25. The binding agent of any one of claims 21-24, comprising a mutant constant region or mutant Fc region genetically engineered to possess enhanced antibody dependent cellular cytotoxicity.

26. The binding agent of any one of claims 21-24, comprising an effectorless constant region or an effectorless Fc domain.

27. The binding agent of any one of claims 1-13, wherein the binding agent is a trispecific antibody comprising the CEACAM5 binding domain, a second antigen binding domain, and a third antigen domain.

28. The binding agent of any one of claims 1-27, wherein the binding agent consists of a single polypeptide chain.

29. The binding agent of any one of claims 1-28, wherein the binding agent consists of two polypeptide chains.

30. The binding agent of claim 29, wherein a first polypeptide chain and a second polypeptide chain are linked by one or more disulfide bonds.

31. The binding agent of claim 29, wherein a first polypeptide chain and a second polypeptide chain are linked by VH-VL pairing.

32. The binding agent of claim 29, wherein each of the first and second polypeptide chains comprises an Fc fragment having complementary mutations to one another that enhance heterodimerization over homodimerization so as to force the association of the first polypeptide chain comprising the CEACAM5 binding domain to a second polypeptide chain comprising the second binding domain.

33. The binding agent of claim 32, wherein the mutations are knob-hole mutations.

34. The binding agent of claim 33, wherein the knob mutation is selected from the group consisting of S354C, T366W and K409A; and wherein the hole mutation is selected from the group consisting of S354C, Y349C, T366S, L368A, F405K, and Y407V.

35. An antibody-drug conjugate (ADC) comprising a cytotoxic drug, a linker, and a binding agent of any one of claims 1-34.

36. The ADC of claim 35, wherein the cytotoxic drug is a maytansinoid, a camptothecin, a dolastatin, an aurastatin, a pyrrolobenzodiazepine (PBD), a calicheamicin, a duocarmycin, a tubulolysin, an analogue thereof, or a combination thereof.

37. The ADC of claim 38, wherein the cytotoxic drug is selected from the group consisting of deruxtecan, exatecan, SN-38, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAE), Auristatin F-hydroxypropylamide, AurOlOl, emtansine (DM1), Maytansinoid DM4, duocarmycin, duostatin-5, and combinations thereof.

38. The ADC of any one of claims 35-37, wherein the linker is a cleavable linker selected from the group consisting of maleimidocaproyl-L-valine-L-citrulline-p-aminobenzoyl carbamate (mc-VC-PABC) and malcimido-dPEG8-L-valinc-L-alalinc- -aminobcnzoyl carbamate (M-dPEG8-VA-PABC).

39. The ADC of any one of claims 35-37, wherein the linker is a non-cleavable linker selected from the group consisting of maleimidocaproyl (MC), succinimidyl-4-(N- maleimidomethyl)cyclohexane-l -carboxylate (SMCC), and thioether-containing linker.

40. The ADC of any one of claims 35-39, wherein the ADC has a drug-to-antibody (DAR) ratio between 2-10.

41. A nucleic acid or set of nucleic acids, which individually or collectively encode the binding agent of any one of claim 1-34.

42. The nucleic acid or the set of nucleic acids of claim 41, encoding a CEACAM5 binding domain comprising a VH region and a VL region, wherein the VH and VL regions are encoded by nucleotide sequences selected from the group consisting of:(a) SEQ ID NOs: 13 and 15, respectively;(b) SEQ ID NOs: 14 and 15, respectively;(c) SEQ ID NOs: 46 and 48, respectively; and(d) SEQ ID NOs: 47 and 48, respectively;43. The nucleic acid or set of nucleic acids of claim 41 or 42, comprising a nucleic acid encoding an anti-CEACAM5 scFv.

44. The nucleic acid or set of nucleic acids of claim 43, wherein the anti- CEACAM5-scFv comprises an amino acid sequence set forth in any one of SEQ ID NOs: 20, 21, 53, or 54.

45. The nucleic acid of set of nucleic acids of any one of claims 41-44, comprising a nucleic acid encoding a self-cleaving 2A peptide from porcine teschovirus-1 (P2A), equine rhinitis A virus (E2A), thosea asigna virus (T2A), or foot-and-mouth disease virus (F2A), or a combination thereof.

46. The nucleic acid or the set of nucleic acids of any one of claims 41-45, which comprise a plasmid.

47. The nucleic acid or the set of nucleic acids of any one of claims 41-46, which comprise an expression vector.

48. The nucleic acid of claim 47, wherein the expression vector is a lentivirus vector.

49. A population of immune cells comprising genetically engineered immune cells expressing or secreting the binding agent of any one of claims 1-34.

50. The population of immune cells of claim 490, wherein the genetically engineered immune cells comprise the nucleic acid or set of nucleic acids of any one of claims 35-48.

51. The population of immune cells of claim 49 or 50, wherein the genetically engineered immune cells are T cells, natural killer (NK) cells, tumor infiltrating lymphocytes, dendritic cells, macrophages, neutrophils, eosinophils, basophils, mast cells, myeloid-derived suppressor cells, stem cells, precursors thereof, subtypes thereof, or a combination thereof; optionally wherein the immune cell is a human immune cell.

52. The population of immune cells of claim 51, wherein the genetically engineered immune cells are T cells.

53. The population of immune cells of claim 52, wherein the T cells express SSTR254. The population of immune cells of claim 51, wherein the genetically engineered immune cells are NK cells.

55. The population of immune cells of claim 54, wherein the NK cells express SSTR2.

56. The population of immune cells of any one of claims 49-55, further comprising a second population of genetically engineered immune cells.

57. The population of immune cells of claim 58, wherein the second population of genetically engineered immune cells expresses a polypeptide of interest.

58. A method of treating cancer, comprising administering to a subject in need thereof an effective amount of the binding agent in any one of claims 1-34.

59. A method of treating cancer, comprising administering to a subject in need thereof an effective amount of the population of immune cells in any one of claims 49-57.

60. The method of claim 58 or 59, wherein the subject is a human patient.

61. The method of any one of claims 58-60, wherein the cancer is a solid tumor.

62. The method of claim 61, wherein the cancer is a carcinoma.

63. The method of claim 62, wherein the carcinoma is a small cell lung carcinoma, non-small cell lung carcinoma, squamous cell lung carcinoma, large cell lung carcinoma, pancreatic carcinoma, pancreatic ductal carcinoma, prostate carcinoma, esophageal carcinoma, breast carcinoma, ovarian carcinoma, prostate carcinoma, colorectal carcinoma, bladder carcinoma, cervical carcinoma, hepatocellular carcinoma, renal hepatocellular carcinoma, gastric carcinoma, papillary carcinoma, adrenocortical carcinoma, pituitary carcinoma, a head and neck carcinoma, an adenocarcinoma thereof, or a squamous cell carcinoma thereof.

64. The method of any one of claims 58-61, wherein the cancer is a glioblastoma or mesothelioma.

65. The method of any one of claims 58-64, wherein the cancer is metastatic.

66. The method of claim 58, further comprising administering a therapy to reduce tumor burden in the subject prior to administration of the binding agent.

67. The method of any one of claims 59-65, further comprising administering a therapy to reduce tumor burden in the subject prior to administering the population of immune cells.

68. The method of claim 66 or 67, wherein the therapy is a chemotherapy, an immunotherapy, a radiotherapy, or a surgery.

69. The method of any one of claims 59-65 or 67, wherein prior to administering the population of cells, the subject received a lymphodepleting treatment to condition the subject for the cell therapy.

70. The method of claim 69, wherein the lymphodepleting treatment comprises administering to the subject one or more of fludarabine and cyclophosphamide.

71. The method of any one of claims 58-65, further comprising administration of an immune checkpoint inhibitor.

72. The method of claim 71, wherein the immune checkpoint inhibitor is pembrolizumab (Keytruda™), ipilimumab (Yervoy™), nivolumab (Opdivo™), or atezolizumab (Tecentriq™).

73. The method of any one of claims 58-65, further comprising administration of a therapeutic antibody selected from the group consisting of abagovomab, adecatumumab, afutuzumab, alemtuzumab, altumomab, amatuximab, anatumomab, arcitumomab, bavituximab, bectumomab, bevacizumab, bivatuzumab, blinatumomab, brentuximab, cantuzumab, catumaxomab, cetuximab, citatuzumab, cixutumumab, clivatuzumab, conatumumab, daratumumab, drozitumab, duligotumab, dusigitumab, detumomab, dacetuzumab, dalotuzumab, ecromeximab, elotuzumab, ensituximab, ertumaxomab, etaracizumab, farietuzumab, ficlatuzumab, figitumumab, flanvotumab, futuximab, ganitumab, gemtuzumab, girentuximab, glembatumumab, ibritumomab, igovomab, imgatuzumab, indatuximab, inotuzumab, intetumumab, ipilimumab, iratumumab, labetuzumab, lexatumumab, lintuzumab, lorvotuzumab, lucatumumab, mapatumumab, matuzumab, milatuzumab, minretumomab, mitumomab, moxetumomab, namatumab, naptumomab, necitumumab, nimotuzumab, nofetumomab, ocaratuzumab, ofatumumab, obinutuzumab, olaratumab, onartuzumab, oportuzumab, oregovomab, panitumumab, parsatuzumab, patritumab, pemtumomab, pertuzumab, pintumomab, pritumumab, racotumomab, radretumab, rilotumumab, rituximab, robatumumab, satumomab, sibrotuzumab, siltuximab, simtuzumab, solitomab, tacatuzumab, taplitumomab, tenatumomab, teprotumumab, tigatuzumab, tositumomab, trastuzumab, tucotuzumab, ublituximab, veltuzumab, vorsetuzumab, votumumab, zalutumumab, CC49 and 3F8.

74. The method of any one of claims 58-65, further comprising administration of a tyrosine kinase inhibitor capable of inhibiting TCR signaling.

75. The method of claim 745, wherein the tyrosine kinase is selected from the group consisting of dasatinib, ponatinib, saracatinib, bosutinib, nilotinib, and combinations thereof.

76. The method of claim 75, wherein the tyrosine kinase inhibitor is dasatinib.

77. The method of any one of claims 74-76, wherein the tyrosine kinase inhibitor is administered for a period of time sufficient to restore at least partial T cell function and then discontinued.

78. The method of any one of claims 74-76, wherein the tyrosine kinase inhibitor is administered continuously.

79. The method of any one of claims 74-76, wherein the tyrosine kinase inhibitor is administered intermittently.

80. The method of claim 79, wherein the tyrosine kinase inhibitor administered iteratively for purposes of facilitating periods of T cell inactivation during pharmaceutical composition administration and periods of T cell activation during absence of pharmaceutical composition administration.

81. The method of claim 80, wherein the tyrosine kinase inhibitor is administered iteratively so that the tyrosine kinase concentration is maintained below a threshold level required to block CAR-T cell function.

82. A method for treating cancer and monitoring distribution of genetically engineered T cells or NK cells in a patient, comprising: incubating the genetically engineered T cells of claim 53 or NK cell of claim 55 with a radioactive label that binds to SSTR2; intravenously infusing the labeled T or NK cells into a patient in an amount of 104-108cells / kg patient; and detecting the labeled T or NK cell distribution by PET / CT imaging, wherein the labeled T or NK cells are infiltrated into cancer cells to kill the cancer cells.

83. The method of claim 82, wherein the label is radioactively labeled DOTATOC or radioactively labeled DOTATATE.

84. The method of claim 83, wherein the DOTATOC or DOTATATE is radiolabeled with68Ga.

85. The method of any one of claims 82-84, wherein the cancer is thyroid cancer, gastric cancer, pancreatic cancer, or breast cancer.

86. A method for treating cancer and monitoring distribution of genetically engineered T cells or NK cells in a patient, comprising: intravenously infusing the population of genetically engineered T cells of claim 53 or NK cell of claim 55 into a patient, wherein the T or NK cells have been transduced to express at least 100,000 molecules of SSTR2 per T or NK cell; injecting into the patient a radioactive label that binds to SSTR2 at least one hour prior to PET / CT imaging; and detecting the labeled T or NK cell distribution by PET / CT imaging, wherein the labeled T or NK cells are infiltrated into cancer cells to kill the cancer cells.

87. The method of claim 86, wherein the cancer is thyroid cancer, gastric cancer, pancreatic cancer, or breast cancer.

88. A method of producing the binding agent in any one of claims 1-34, comprising the steps of: introducing one or more expression vectors encoding the CEACAM5 binding agent; culturing the cells under conditions sufficient for expressing one or two polypeptide chains of the CEACAM5 binding agent; and harvesting the binding agent produced from the cultured cells.

89. The method of claim 78, further comprising the step of subjecting the one or two polypeptide chains to reducing conditions sufficient for forming disulfide bonds between the one or two polypeptide chains, thereby producing the binding agent.

90. A method of producing a population of genetically engineered immune cells, the method comprising: introducing into a population of immune cells a nucleic acid or set of nucleic acids coding for the binding agent of any one of claims 1-34 to produce a population of genetically engineered immune cells.

91. The method of claim 90, wherein the population of genetically engineered immune cells are T cells, natural killer cells, tumor infiltrating lymphocytes, dendritic cells, macrographs, neutrophils, eosinophils, basophils, mast cells, myeloid-derived suppressor cells, stem cells, precursors thereof, or a combination thereof; optionally wherein the immune cell is a human immune cell.

92. The method of claim 91, wherein the population of genetically engineered immune cells are T cells.

93. The method of claim 92, comprising expanding the population of T cells in the presence of IL-2 and / or a tyrosine kinase inhibitor capable of inhibiting TCR signaling and / or CAR signaling.

94. The method of claim 93, wherein the tyrosine kinase is dasatinib, ponatinib, saracatinib, bosutinib, nilotinib, or a combination thereof.

95. The method of claim 94, wherein the tyrosine kinase is dasatinib.

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