Methods of treating EGFR mutant cancers

Abstract

The present disclosure provides methods of treating a cancer in subject in need thereof comprising administering a bispecific gamma delta (γδ)-T cell engager (BS-GDTE) comprising a first antigen-binding domain that specifically binds to epidermal growth factor receptor (EGFR) and a second antigen-binding domain that binds to human Vδ2, wherein the cancer expresses a mutant EGFR.

Description

METHODS OF TREATING EGFR MUTANT CANCERSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Application 63 / 611 ,683, filed December 18, 2023, the contents of which are incorporated herein by reference in its entirety.SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (SEAG_001_01WO_SeqList_ST26.xml; Size: 37,710 bytes; and Date of Creation: December 9, 2024) are herein incorporated by reference in its entirety.BACKGROUND

[0003] EGFR is a 170 kDa glycoprotein that consists of an extracellular receptor domain, a transmembrane region, and an intracellular domain with tyrosine kinase function. It is a receptor for members of the epidermal growth factor family of extracellular protein ligands such as epidermal growth factor (EGF), transforming growth factor alpha (TGF-a) or heparin- binding EGF-like growth factor. Under physiological conditions, the receptor is widely expressed in a number of tissues such as in the thyroid gland, skin, lung, colon, liver, kidney, or placenta tissues.

[0004] The EGFR and its downstream signaling pathways play key roles in oncogenesis: overexpression because of gene amplification and mutations affecting EGFR expression and / or activity can contribute to cancer due to over-activation or constitutive activation of downstream signaling pathways that lead to uncontrolled cell proliferation and evasion of apoptosis. EGFR targeting therapies have been developed, including receptor blocking monoclonal antibodies (mAb), e.g., cetuximab, panitumumab, and necitumumab.

[0005] Although existing anti-EGFR therapies provide significant survival benefit to patients, the duration of response is transient as patients develop secondary resistance, for example by escape mutations in the EGFR extracellular domain (ECD). Thus, there remains a need in the art for compositions and methods for the treatment of cancers expressing a mutant EGFR.SUMMARY

[0006] In some embodiments, the present disclosure provides a method of treating a cancer in subject in need thereof comprising administering a bispecific gamma delta (yb)-T cell engager (BS-GDTE) comprising a first antigen-binding domain that specifically binds to epidermal growth factor receptor (EGFR) and a second antigen-binding domain that binds to human V52, wherein the cancer expresses a mutant EGFR.

[0007] In some embodiments, the mutant EGFR comprises one or more amino acid mutations at a position selected from F404, V441 , S442, R451 , S464, G465, K467, 1491 , and S492 relative to SEQ ID NO: 11. In some embodiments, the mutant EGFR comprises an F404Vmutation. In some embodiments, the mutant EGFR comprises a mutation selected from V441G, V441 D, V441 F, and V441 I. In some embodiments, the mutant EGFR comprises a mutation selected from S442R and S442N. In some embodiments, the mutant EGFR comprises an R451C mutation. In some embodiments, the mutant EGFR comprises an S464L mutation. In some embodiments, the mutant EGFR comprises a mutation selected from G465R, G465E, and G465V. In some embodiments, the mutant EGFR comprises a K467T mutation. In some embodiments, the mutant EGFR comprises a mutation selected from 1491 M and 1491 R In some embodiments, the mutant EGFR comprises an S492R mutation.

[0008] In some embodiments, the cancer is selected from colorectal cancer (CRC), head and neck squamous cell cancer (HNSCC), non-small cell lung cancer (NSCLC), pancreatic ductal adenocarcinoma (PDAC), clear cell renal carcinoma, papillary cell renal carcinoma, urothelial cancer, bladder cancer, and breast cancer. In some embodiments, the cancer is selected from colorectal cancer (CRC), head and neck squamous cell cancer (HNSCC), non-small cell lung cancer (NSCLC), and pancreatic ductal adenocarcinoma (PDAC).

[0009] In some embodiments, the first antigen-binding domain of the BS-GDTE comprises a CDR1 of SEQ ID NO: 6, a CDR2 of SEQ ID NO: 7, and a CDR3 of SEQ ID NO: 8, and wherein the second antigen-binding domain of the BS-GDTE comprises a CDR1 of SEQ ID NO: 1 , a CDR2 of SEQ ID NO: 2, and a CDR3 of SEQ ID NO: 3. In some embodiments, the first antigenbinding domain is a VHH domain comprising SEQ ID NO: 9 and wherein the second antigenbinding domain is a VHH domain comprising SEQ ID NO: 4 In some embodiments, the BS- GDTE comprises the amino acid sequence of SEQ ID NO: 5 and SEQ ID NO: 10.

[0010] In some embodiments, the present disclosure provides a method of killing a tumor cell that expresses a mutant epidermal growth factor receptor (EGFR) comprising exposing the tumor cell to a BS-GDTE comprising the amino acid sequence of SEQ ID NO: 5 and SEQ ID NO: 10.

[0011] In some embodiments, the mutant EGFR comprises one or more amino acid mutations at a position selected from F404, V441 , S442, R451 , S464, G465, K467, 1491 , and S492 relative to SEQ ID NO: 11. In some embodiments, the mutant EGFR comprises an F404V mutation. In some embodiments, the mutant EGFR comprises a mutation selected from V441G, V441 D, V441 F, and V441 I. In some embodiments, the mutant EGFR comprises a mutation selected from S442R and S442N. In some embodiments, the mutant EGFR comprises an R451C mutation. In some embodiments, the mutant EGFR comprises an S464L mutation. In some embodiments, the mutant EGFR comprises a mutation selected from G465R, G465E, and G465V. In some embodiments, the mutant EGFR comprises a K467T mutation. In some embodiments, the mutant EGFR comprises a mutation selected from 1491 M and 1491 R In some embodiments, the mutant EGFR comprises an S492R mutation.

[0012] In some embodiments, the tumor cell is in vitro, ex vivo, or in vivo.

[0013] In some embodiments, the present disclosure provides a method of treating a cancer in subject in need thereof comprising administering a bispecific gamma delta (Y6)-T cell engager (BS-GDTE) comprising a first antigen-binding domain that specifically binds to epidermal growth factor receptor (EGFR) and a second antigen-binding domain that binds to human V52, wherein the cancer is resistant to one or more EGFR-targeted therapies. In some embodiments, the EGFR-targeted therapy is a small molecule, a monoclonal antibody, or an antibody prodrug. In some embodiments, the small molecule is selected from gefitnib and Osimertinib. In some embodiments, the monoclonal antibody is selected from cetuximab and panitumumab. In some embodiments, the antibody prodrug is CX-904.

[0014] In some embodiments, the cancer expresses a mutant EGFR. In some embodiments, the cancer expresses a mutant EGFR selected from S492R, G465R, G465E, S464L, K467T, and V441 D and is resistant to treatment with cetuximab. In some embodiments, the cancer expresses a mutant EGFR selected from G465R, G465E, and S464L and is resistant to treatment with panitumumab.

[0015] In some embodiments, the first antigen-binding domain of the BS-GDTE comprises a CDR1 of SEQ ID NO: 6, a CDR2 of SEQ ID NO: 7, and a CDR3 of SEQ ID NO: 8, and wherein the second antigen-binding domain of the BS-GDTE comprises a CDR1 of SEQ ID NO: 1 , a CDR2 of SEQ ID NO: 2, and a CDR3 of SEQ ID NO: 3. In some embodiments, the first antigenbinding domain is a VHH domain comprising SEQ ID NO: 9 and wherein the second antigenbinding domain is a VHH domain comprising SEQ ID NO: 4 In some embodiments, the BS- GDTE comprises the amino acid sequence of SEQ ID NO: 5 and SEQ ID NO: 10BRIEF DESCRIPTION OF THE FIGURES

[0016] Fig. 1 provides histograms of SGN-EGFRd2 binding on various EGFR ECD mutations as compared to Cetuximab, Panitumab, and isotype controls. Geometric mean fluorescence intensities of each antibody are depicted on each A2058 cell line engineered to express a specific EGFR variant.

[0017] Fig. 2A - Fig. 2H provide sensorgrams depicting shift during association and dissociation of SGN-EGFR2 to various EGFRd2 ECD mutations (Fig. 2A - WT EGFR, Fig. 2B - EGFR V441 D, Fig. 2C - EGFR S492R, Fig. 2D - EGFR G465R, Fig. 2E - EGFR G465E, Fig. 2F - EGFR F404V, Fig. 2G - EGFR S442R, Fig. 2H - EGFR G465V).

[0018] Fig. 3A - Fig. 3B illustrate degranulation (Fig. 3A) and IFNy (Fig. 3B) responses of y<5 T cells after co-culture with EGFR-expressing A2058 cells.DETAILED DESCRIPTION

[0019] SGN-EGFRd2 is bispecific gamma delta (yb)-T cell engager (BS-GDTE) that targets EGFR. SGN-EGFRd2 comprises humanized variable heavy chain (VHH) domains against Vy9V<52 (gamma delta) T cells and epidermal growth factor receptor (EGFR). Anti-tumoractivity occurs when the bispecific SGN-EGFRd2 engages EGFR expressing tumor cells with gamma delta T cells. The crosslinked EGFR-positive tumor cells expressing butyrophilins, when in complex with phosphoantigens, activate Vy9V<52 T cells to direct tumor cell killing by degranulation and secretion of cytolytic molecules. Amino acid sequences of SGN-EGFRd2 are provided in Table 1. The full SGN-EGFRd2 agent is a heterodimer of SEQ ID NO: 5 and SEQ ID NO: 10.Table 1 : SGN-EGFRd2 amino acid sequences

[0020] In some embodiments, the present disclosure provides a method of treating a cancer in subject in need thereof comprising administering a bispecific gamma delta (yb)-T cell engager (BS-GDTE) (e.g., SGN-EGFRd2) comprising a first antigen-binding domain that specifically binds to epidermal growth factor receptor (EGFR) and a second antigen-binding domain that binds to human V52, wherein the cancer expresses a mutant EGFR.

[0021] EGFR is an oncogenic driver and target of several classes of therapeutic modalities, including antibodies. Two anti-EGFR antibodies are currently approved for treatment of KRAS wild type metastatic colorectal cancer (mCRC), Erbitux® (cetuximab) and Vectibix® (panitumumab). Although these anti-EGFR therapies provide significant survival benefit to patients, duration of response is transient as most patients develop secondary resistance, for example by escape mutations in the EGFR extracellular domain (ECD). Various point mutations in the EGFR ECD have been identified as escape variants from existing anti-EGFR therapies, including mutations at positions F404, V441 , S442, R451 , S464, G465, K467, 1491 , and S492.

[0022] The wildtype amino acid sequence of the EGFR ECD is provided in SEQ ID NO: 11. In some embodiments, the mutant EGFR comprises one or more amino acid mutations at a position selected from F404, V441 , S442, R451 , S464, G465, K467, 1491 , and S492 relative to SEQ ID NO: 11.

[0023] In some embodiments, the mutant EGFR comprises an F404V mutation. In some embodiments, the mutant EGFR comprises a mutation selected from V441G, V441 D, V441 F, and V441 I. In some embodiments, the mutant EGFR comprises a mutation selected from S442R and S442N. In some embodiments, the mutant EGFR comprises an R451C mutation. In some embodiments, the mutant EGFR comprises an S464L mutation. In some embodiments, the mutant EGFR comprises a mutation selected from G465R, G465E, and G465V. In some embodiments, the mutant EGFR comprises a K467T mutation. In some embodiments, the mutant EGFR comprises a mutation selected from 1491 M and 1491 R. In some embodiments, the mutant EGFR comprises a mutation selected from S492Rand S492N.

[0024] In some embodiments, the mutant EGFR comprises a V441 D mutation. In some embodiments, the mutant EGFR comprises a V441 F mutation. In some embodiments, the mutant EGFR comprises an S442R mutation. In some embodiments, the mutant EGFR comprises an S464L mutation. In some embodiments, the mutant EGFR comprises a G465R mutation. In some embodiments, the mutant EGFR comprises a G465E mutation. In some embodiments, the mutant EGFR comprises a G465V mutation. In some embodiments, the mutant EGFR comprises a 1491 R mutation. In some embodiments, the mutant EGFR comprises a S492R mutation.

[0025] In some embodiments, the present disclosure provides methods of treating cancer comprising administering a BS-GDTE comprising a first antigen-binding domain that specifically binds to epidermal growth factor receptor (EGFR) and a second antigen-binding domain that binds to human V52, wherein the cancer is resistant to one or more EGFR- targeted therapies. In some embodiments, the cancer is resistant to treatment with one or more EGFR-targeted therapies. In some embodiments, the cancer is refractory to treatment with one or more EGFR-targeted therapies.

[0026] In some embodiments, the EGFR-targeted therapy is selected from a small molecule (e.g., gefitnib, Osimertinib), a monoclonal antibody (e.g., cetuximab, panitumumab), or an antibody prodrug (e.g., Probody such as CX-904). In some embodiments, the cancer expresses a mutant EGFR selected from S492R, G465R, G465E, G465V, S464L, K467T, and V441 D and is resistant to treatment with cetuximab. In some embodiments, the cancer expresses a mutant EGFR selected from G465R, G465E, G465V, S442R, and S464L and is resistant to treatment with panitumumab. In some embodiments, the cancer expresses a mutant EGFR selected from G465E, G465R, G465V, S442R, S464L, S492R, V441 D, and V441 F and is resistant to treatment with cetuximab. In some embodiments, the cancer expresses a mutant EGFR selected from G465E, G465R, G465V, 1491 R, S442R, and S464L and is resistant to treatment with panitumumab.

[0027] In some embodiments, the cancer comprising the mutant EGFR is selected from colorectal cancer (CRC), head and neck squamous cell cancer (HNSCC), non-small cell lung cancer (NSCLC), pancreatic ductal adenocarcinoma (PDAC), clear cell renal carcinoma, papillary cell renal carcinoma, urothelial cancer, bladder cancer, and breast cancer. In some embodiments, the cancer is colorectal cancer (CRC). In some embodiments, the cancer is head and neck squamous cell cancer (HNSCC). In some embodiments, the cancer is non- small cell lung cancer (NSCLC). In some embodiments, the cancer is pancreatic ductal adenocarcinoma (PDAC). In some embodiments, the cancer is clear cell renal carcinoma. In some embodiments, the cancer is papillary cell renal carcinoma. In some embodiments, the cancer is urothelial cancer. In some embodiments, the cancer is bladder cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer comprising the mutant EGFR is selected from CRC, HNSCC, NSCLC, and PDAC. For example, the prevalence of the S492R mutation in CRC is 16.1%. Prevalence of additional mutations are as follows: G465R - 16%, G465E - 14%, S464L - 14%, V441G - 7%, V441 D - 6%, V441 F - 5-8%, V441 I - 5-8%.

[0028] Administration of SGN-EGFRd2 can occur by injection, irrigation, inhalation, consumption, electro-osmosis, hemodialysis, iontophoresis, and other methods known in the art. In some embodiments, administration route is local or systemic. In some embodiments administration route is intraarterial, intracranial, intradermal, intraduodenal, intrammamary, intrameningeal, intraperitoneal, intrathecal, intratumoral, intravenous, intravitreal, ophthalmic, parenteral, spinal, subcutaneous, ureteral, urethral, vaginal, or intrauterine.

[0029] In some embodiments, the administration route is by infusion (e.g., continuous or bolus). Examples of methods for local administration, that is, delivery to the site of injury or disease, include through an Ommaya reservoir, e.g. for intrathecal delivery (See e.g., US Patent Nos. 5,222,982 and 5,385,582, incorporated herein by reference); by bolus injection, e.g. by a syringe, e.g. into a joint; by continuous infusion, e.g. by cannulation, such as withconvection. In some embodiments, the administration route is by topical administration or direct injection.

[0030] In some embodiments, SGN-EGFRd2 is administered to a subject in order to treat a cancer expressing a mutant EGFR. In some embodiments, treatment comprises delivering an effective amount of SGN-EGFRd2 or composition thereof to a subject in need thereof. In some embodiments, treating refers to the treatment of a disease in a mammal, e.g., in a human, including (a) inhibiting the disease, i.e., arresting disease development or preventing disease progression; (b) relieving the disease, i.e., causing regression of the disease state or relieving one or more symptoms of the disease; and (c) curing the disease, i.e., remission of one or more disease symptoms. In some embodiments, treatment may refer to a short-term (e.g., temporary and / or acute) and / or a long-term (e.g., sustained) reduction in one or more disease symptoms. In some embodiments, treatment results in an improvement or remediation of the symptoms of the disease. The improvement is an observable or measurable improvement or may be an improvement in the general feeling of well-being of the subject.

[0031] The effective amount of SGN-EGFRd2 administered to a particular subject will depend on a variety of factors, several of which will differ from patient to patient including the disorder being treated and the severity of the disorder; activity of the specific agent(s) employed; the age, body weight, general health, sex and diet of the patient; the timing of administration, route of administration; the duration of the treatment; drugs used in combination; the judgment of the prescribing physician; and like factors known in the medical arts.

[0032] In some embodiments, the BS-GDTE (e.g., SGN-EGFRd2) is administered to a subject at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, or more times.

[0033] The term “composition” as used herein refers to a formulation of SGN-EGFRd2 that is capable of being administered or delivered to a subject or cell. Typically, formulations include all pharmaceutically acceptable compositions including derivatives and / or prodrugs, solvates, stereoisomers, racemates, or tautomers thereof with any physiologically acceptable carriers, diluents, and / or excipients.

[0034] The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0035] As used herein “pharmaceutically acceptable carrier, diluent or excipient” includes without limitation any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, surfactant, and / or emulsifier which hasbeen approved by the United States Food and Drug Administration as being acceptable for use in humans and / or domestic animals. Exemplary pharmaceutically acceptable carriers include, but are not limited to, to sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; tragacanth; malt; gelatin; talc; cocoa butter, waxes, animal and vegetable fats, paraffins, silicones, bentonites, silicic acid, zinc oxide; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen- free water; isotonic saline; Ringer’s solution; ethyl alcohol; phosphate buffer solutions; and any other compatible substances employed in pharmaceutical formulations. Except insofar as any conventional media and / or agent is incompatible with the agents of the present disclosure, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions.

[0036] “Pharmaceutically acceptable salt” includes both acid and base addition salts. Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1 ,2- disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1 ,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, ptoluenesulfonic acid, trifluoroacetic acid, undecylenic acid, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as ammonia,isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.

[0037] Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

[0038] Examples of pharmaceutically-acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0039] Further guidance regarding formulations that are suitable for various types of administration can be found in Remington’s Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, Pa., 17th ed. (1985). For a brief review of methods for drug delivery, see, Langer, Science 249:1527-1533 (1990).EXAMPLESExample 1

[0040] To understand the binding profile of anti-EGFR antibodies on EGFR ECD, EGFR negative A2058 cells were transduced to express EGFR ECD variants (N=17), including wild type EGFR. The amino acid sequences of the EGFR mutants are provided below in Table A. The signal peptide sequence, which is cleaved from the mature protein, is shown in bold italicized text.Table A - EGFR Amino Acid sequences

[0041] The binding specificity of anti-EGFR antibodies then evaluated by flow cytometry. A2058 cell lines were lifted with Versene, resuspended in cold FACS buffer (2% FBS + 0.02% Sodium Azide in PBS) and plated at 100,000 cells / well in a 96 well V bottom plate. Cells werestained by incubation with 1 pg / mL final primary antibody concentration for 30 minutes on ice. Samples were spun down and washed with cold FACS buffer twice. Samples were then incubated in FACS buffer containing fluorescently labeled secondary antibody for 30 minutes on ice and devoid of light before spun down and washed twice with ice cold FACS buffer. Sample were then analyzed on an Attune Flow Cytometer (Invitrogen). FlowJo software 10.8.1 was used to analyze the data. As shown in Fig. 1 , binding of SGN-EGFRd2 was agnostic to all identified EGFR ECD mutants whereas cetuximab and panitumumab binding displayed differential binding, suggesting ablation of the epitope.

[0042] Binding kinetics of SGN-EGFRd2 and anti-EGFR therapeutic molecules Cetuximab (Erbitux®, Eli Lilly) and Panitumumab (Vectibix®, Amgen) were also assessed on recombinant EGFR ECD mutant proteins. The antibodies were diluted in kinetic buffer (0.5% BSA + 0.2% Tween20, 1x PBS pH 7.4) and loaded onto anti-human Fc (AHC) biosensors (Sartorius) at 4 pg / mL for 300 seconds. After baselining in kinetic buffer, serial dilutions of recombinant EGFR proteins (0.5, 2.4, 11.2, 51 , 236, 1087, and 5000 nM or 9.7, 33, 112, 382, 1298, 4412, and 15,000 nM) in kinetic buffer were allowed to associate with antibody immobilized on biosensors until the top concentration of recombinant protein reached equilibrium with antibody (100 or 300 seconds). Then, biosensors were incubated in kinetic buffer to allow for recombinant EGFR protein dissociation to occur (500 or 1000 seconds). Sensorgrams capturing the association and dissociation of recombinant protein from antibody were generated at 30° C on an Octet HTX system (Sartorius). Reference biosensors with immobilized antibody were measured in the absence of recombinant protein. Negative control biosensors without immobilized antibody were assessed with recombinant protein present at 4412, 5000, and 15,000 nM to verify the absence of nonspecific binding of the recombinant proteins to the AHC biosensors themselves. As show in Fig. 2A - Fig. 2H, analysis of binding affinity as ratio of association and dissociation on EGFR ECD variants shows that SGN- EGFRd2 binding is not abrogated across the panel of mutations.Example 2

[0043] In this study, activation and cytolytic activity of SGN-EGFRd2 was assessed on select EGFR ECD mutants. A2058 tumor cells expressing EGFR ECD variants were labeled using 1 pM of CFSE (Invitrogen) are plated at 10,000 cells / well in a 96 flat well plate. 10,000 zoledronate-expanded y<5 T cells were added to the plate after an overnight rest in in 50 U / rnL of IL-2. A titration of y<5 T cell engagers was also plated. Brefeldin A (Biolegend) and 5 pg / mL of BV605 anti-human CD 107a antibody was added to the mix. The coculture was placed in an incubator for 4 hours at 37°C. To ensure all cells were removed from the plate, the cells were quickly lifted using Accutase (Invitrogen) and washed in staining buffer (eBioscience). The cultured was stained with LIVE / DEAD™ fixable Aqua Dead Cell Stain Kit (Invitrogen), anti-CD3 BV711 (Biolegend), anti-TCR Vy9 PE-cy7 (Biolegend), and anti-IFNy BV421 (Biolegend) and analyzed on a Attune Flow Cytometer (Invitrogen). Flowjo software 10.7.1 was used to analyze the data. As shown in Fig. 3A and Fig. 3B, activation and cytolytic activity of SGN- EGFRd2 was retained on EGFR ECD mutants.

Claims

CLAIMS1. A method of treating a cancer in subject in need thereof comprising administering a bispecific gamma delta (yb)-T cell engager (BS-GDTE) comprising a first antigen-binding domain that specifically binds to epidermal growth factor receptor (EGFR) and a second antigen-binding domain that binds to human V52, wherein the cancer expresses a mutant EGFR.

2. The method of claim 1 , wherein the mutant EGFR comprises one or more amino acid mutations at a position selected from F404, V441 , S442, R451 , S464, G465, K467, 1491 , and S492 relative to SEQ ID NO: 11.

3. The method of claim 2, wherein the mutant EGFR comprises an F404V mutation.

4. The method of claim 2, wherein the mutant EGFR comprises a mutation selected from V441G, V441 D, V441 F, and V441 I.

5. The method of claim 2, wherein the mutant EGFR comprises a mutation selected from S442R and S442N.

6. The method of claim 2, wherein the mutant EGFR comprises an R451C mutation.

7. The method of claim 2, wherein the mutant EGFR comprises an S464L mutation.

8. The method of claim 2, wherein the mutant EGFR comprises a mutation selected from G465R, G465E, and G465V.

9. The method of claim 2, wherein the mutant EGFR comprises a K467T mutation.

10. The method of claim 2, wherein the mutant EGFR comprises a mutation selected from I491M and I491 R11 . The method of claim 2, wherein the mutant EGFR comprises an S492R mutation.

12. The method of any one of claims 1-11 , wherein the cancer is selected from colorectal cancer (CRC), head and neck squamous cell cancer (HNSCC), non-small cell lung cancer (NSCLC), pancreatic ductal adenocarcinoma (PDAC), clear cell renal carcinoma, papillary cell renal carcinoma, urothelial cancer, bladder cancer, and breast cancer.

13. The method of any one of claims 1-11 , wherein the cancer is selected from colorectal cancer (CRC), head and neck squamous cell cancer (HNSCC), non-small cell lung cancer (NSCLC), and pancreatic ductal adenocarcinoma (PDAC).

14. The method of any one of claims 1-13, wherein the first antigen-binding domain of the BS-GDTE comprises a CDR1 of SEQ ID NO: 6, a CDR2 of SEQ ID NO: 7, and a CDR3 of SEQ ID NO: 8, and wherein the second antigen-binding domain of the BS-GDTE comprises a CDR1 of SEQ ID NO: 1 , a CDR2 of SEQ ID NO: 2, and a CDR3 of SEQ ID NO: 3.

15. The method of claim 14, wherein the first antigen-binding domain is a VHH domain comprising SEQ ID NO: 9 and wherein the second antigen-binding domain is a VHH domain comprising SEQ ID NO:

416. The method of claim 14 or 15, wherein the BS-GDTE comprises the amino acid sequence of SEQ ID NO: 5 and SEQ ID NO: 10.

17. A method of killing a tumor cell that expresses a mutant epidermal growth factor receptor (EGFR) comprising exposing the tumor cell to a BS-GDTE comprising the amino acid sequence of SEQ ID NO: 5 and SEQ ID NO: 10.

18. The method of claim 17, wherein the mutant EGFR comprises one or more amino acid mutations at a position selected from F404, V441 , S442, R451 , S464, G465, K467, 1491 , and S492 relative to SEQ ID NO: 11.

19. The method of claim 18, wherein the mutant EGFR comprises an F404V mutation.

20. The method of claim 18, wherein the mutant EGFR comprises a mutation selected from V441G, V441 D, V441 F, and V441 I.

21. The method of claim 18, wherein the mutant EGFR comprises a mutation selected from S442R and S442N.

22. The method of claim 18, wherein the mutant EGFR comprises an R451C mutation.

23. The method of claim 18, wherein the mutant EGFR comprises an S464L mutation.

24. The method of claim 18, wherein the mutant EGFR comprises a mutation selected from G465R, G465E, and G465V.

25. The method of claim 18, wherein the mutant EGFR comprises a K467T mutation.

26. The method of claim 18, wherein the mutant EGFR comprises a mutation selected from 1491 M and 1491 R27. The method of claim 18, wherein the mutant EGFR comprises an S492R mutation.

28. The method of any one of claims 17-27, wherein the tumor cell is in vitro, ex vivo, or in vivo.

29. A method of treating a cancer in subject in need thereof comprising administering a bispecific gamma delta (yb)-T cell engager (BS-GDTE) comprising a first antigen-binding domain that specifically binds to epidermal growth factor receptor (EGFR) and a second antigen-binding domain that binds to human V52, wherein the cancer is resistant to one or more EGFR-targeted therapies.

30. The method of claim 29, wherein the EGFR-targeted therapy is a small molecule, a monoclonal antibody, or an antibody prodrug.

31. The method of claim 30, wherein the small molecule is selected from gefitnib and Osimertinib.

32. The method of claim 30, wherein the monoclonal antibody is selected from cetuximab and panitumumab.

33. The method of claim 30, wherein the antibody prodrug is CX-904.

34. The method of any one of claims 29-33, wherein the cancer expresses a mutant EGFR.

35. The method of claim 34, wherein the cancer expresses a mutant EGFR selected from S492R, G465R, G465E, G465V, S464L, K467T, and V441 D and is resistant to treatment with cetuximab.

36. The method of claim 34, wherein the cancer expresses a mutant EGFR selected from G465R, G465E, G465V, S442R and S464L and is resistant to treatment with panitumumab.

37. The method of any one of claims 29-36, wherein the first antigen-binding domain of the BS-GDTE comprises a CDR1 of SEQ ID NO: 6, a CDR2 of SEQ ID NO: 7, and a CDR3 of SEQ ID NO: 8, and wherein the second antigen-binding domain of the BS-GDTE comprises a CDR1 of SEQ ID NO: 1 , a CDR2 of SEQ ID NO: 2, and a CDR3 of SEQ ID NO: 3.

38. The method of claim 37, wherein the first antigen-binding domain is a VHH domain comprising SEQ ID NO: 9 and wherein the second antigen-binding domain is a VHH domain comprising SEQ ID NO:

439. The method of claim 37 or 38, wherein the BS-GDTE comprises the amino acid sequence of SEQ ID NO: 5 and SEQ ID NO: 10.

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