Antibody directed enzyme pro-drug therapy (ADEPT) for cancer treatment using an antibody or fragment that binds to a CD206 receptor and 3-cyclopentyl-alpha-phenylalanine-methotrexate

Abstract

Disclosed are methods of treating cancer by administering a first agent comprising an antibody or fragment thereof that binds CD206 present on tumor associated macrophages conjugate to a mutant of human carboxypeptidase A (hCPA), followed by administering second agent comprising 3-cyclopentyl-alpha-phenylalanine-methotrexate.

Description

VIA EFS Attorney Docket No. 044797.00005 Date of Deposit: December 22, 2025ANTIBODY DIRECTED ENZYME PRO-DRUG THERAPY (ADEPT) FOR CANCER TREATMENT USING AN ANTIBODY OR FRAGMENT THAT BINDS TO A CD206 RECEPTOR AND 3-CYCLOPENTYL-ALPHA-PHENYLALANINE-METHOTREXATECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority under 35 U. S. C. § 119(e) to U. S. Provisional Application No: 63 / 738,342, filed December 23, 2024, which is incorporated herein by reference in its entirety.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on November 18, 2025, is named 044797.00005_ST. xml and is 42 KB in size.BACKGROUND OF THE DISCLOSURE

[0003] Glioblastoma is a malignant and pervasive subtype of glioma and is the most common primary brain tumor in adults. The incidence rate of glioblastoma is 3.2 per 100,000 in the adult population and 0.85 per 100,000 in the pediatric population. Glioblastoma is an incurable disease with a median survival of 15 months. Surgical resection, followed by chemotherapy, administered systemically as an active chemical agent, is the gold standard treatment for glioblastoma. Because it is administered systemically, the entire body is exposed to the active chemotherapy agent, and while they greatly affect rapidly dividing cells (i.e., cancerous cells), detrimental side effects on healthy cells are a known drawback (Grochans et al., Cancers (Basel). 14(10):2412, pp. 1-32 (2022); Kaina, J. Clin. Med. 12(23):7442, pp. 1-26 (2023)). Removal of the entire tumor, including microsatellite tumors, without damaging the brain, is difficult by surgical resection. Thus, further treatment is most often required after surgical resection. (Davis, Clin. J. Oncol. Nurs. 20(5):S2-8 (2016); Minniti et al., Radiat. Oncol. 16(1):36 (2021) Pichardo-Rojas et al., J. Neurooncol.169(3):469-487 (2024)).

[0004] Antibody directed enzyme pro-drug therapy (ADEPT) offers a possibility for circumventing these adverse side effects, by administering an inactive pro-drug that will not be exposed to the entire body. Instead, the inactive pro-drug does not become activated into the toxic drug until it reaches and becomes attached to the tumor. The ADEPT system also includes an antibody-enzyme conjugate, wherein the antibody directly targets an antigen present on a cancer 1AFSDOCS:304317774.1VIA EFS Attorney Docket No. 044797.00005 Date of Deposit: December 22, 2025cell. The enzyme cleaves the pro-drug into an active form. Theoretically, the pro-drug can be delivered systemically without causing the unwanted side effects associated with systemic drug delivery.

[0005] The concept of ADEPT therapy has been around since the 1980s. The first small-scale clinical trial was performed by a group at the Charing Cross Hospital in London (Bagshawe et al., Dis. Markers 9(3-4):233-8 (1991)). There have been at least four small clinical trials based an ADEPT approach. However, none have successfully been approved by a regulatory body (Sharma and Bagshawe, Expert Opin. Biol. Ther. 17(1):1-13 (2017)).

[0006] Previous trials have failed mainly due to two problems: 1) the inability to identify an appropriate cancer-specific antigen for purposes of targeting the antibody; and 2) an effective enzyme and pro-drug combination that allows for tumor-specific activation of the pro-drug. Previous attempts at ADEPT have encountered toxicity and reactivity problems. Toxicity occurred from long half-lives of activated drugs, which diffused back into circulation (Springer et al., Cell Biophys. 22(1-3).9-26 (1993) and Martin et al., Cancer Chemother. Pharmacol. 40(3): 189-201 (1997)) or from a long antibody half-life, which required additional antibody clearance (Napier et al., Clin. Cancer Res. 6(3):765-72 (2000)). ADEPT therapy has been further hindered by adverse immune reactions due to the sources of the antibody (often murine) and enzyme (often bacterial) (Bagshawe and Sharma, Transplant. Proc. 28(6):3156-8 (1996)).

[0007] Identifying tumor-specific targets has proven especially difficult. Cancer cells are generally abnormal versions of a patient’s regular, non-cancerous cells. For example, in glioblastomas, cancerous glial cells grow abnormally due to mutations that affect cell cycle checkpoints and the apoptotic pathway, causing them to proliferate at abnormal rates. However, these mutations simply render normal cellular factors unable to perform their expected role, and do not necessarily provide a novel antigen on a cancer cell targetable by ADEPT that is not present on healthy glial cells. At best, some targets have been found that may have altered expression levels between normal cells and their mutated tumorigenic versions, but factors that are specifically expressed by cancer cells remain elusive. Another major difficulty in identifying tumor-specific antigens lies in the heterogeneity of cancer cells within a tumor. This obstacle has been further complicated by the variability in mutations between patients - because no two tumors are alike, a cancer antigen target that works well for one patient may not work in another. Thus, even if or2AFSDOCS:304317774.1VIA EFS Attorney Docket No. 044797.00005 Date of Deposit: December 22, 2025when viable antigenic targets can be found on tumor cells that are specific enough to avoid targeting normal, healthy cells, these are likely to differ from patient to patient.

[0008] Furthermore, the pro-drug must be selectively activated by the ADEPT enzyme. In addition, it must be stable and not susceptible to activation by endogenous enzymes. Use of naturally occurring human enzymes risks activation of the pro-drug in any tissue in which the endogenous enzyme naturally resides. If a foreign enzyme is used, then the immune system will produce anti-enzyme antibodies, as occurred in the early clinical trials (Bagshawe and Sharma, Transplant. Proc. 28(6):3156-8 (1996)). Finally, to be effective, the enzyme must remain stably bound to the antibody long enough for the antibody to bind and target the tumor, for unbound antibodies to be cleared by the body, and for the pro-drug to subsequently be administered and encounter the enzyme at the tumor site.

[0009] A need remains in clinical oncology for an ADEPT system that is improved from one or more of these standpoints.SUMMARY OF THE DISCLOSURE

[0010] The presently disclosed methods and systems of treating cancer with an antibody-enzyme conjugate and a pro-drug are expected to address these needs. A first aspect of the present disclosure is directed to a method of treating cancer characterized by the presence of a solid tumor, comprising administering to a human patient in need thereof a first agent comprising an antibody or fragment thereof that binds a CD206 receptor (mannose macrophage receptor; MMR) present on tumor associated macrophages (TAMs), conjugated to a mutant of human carboxypeptidase A (hCPA) wherein the mutant contains a glycine residue at position 268 in place of threonine (hCPA T268G); followed by administering to the patient a second agent comprising 3-cyclopentyl-alpha- phenylalanine-methotrexate.

[0011] Another aspect of the present disclosure is directed to a system for treating a cancer characterized by the presence of a solid tumor. The system includes a) a first agent comprising an antibody or fragment thereof that binds a CD206 receptor (mannose macrophage receptor; MMR) present on tumor associated macrophages (TAMs), conjugated to a point mutant version of human carboxypeptidase A (hCPA) wherein the mutant contains a glycine residue at position 268 in place of threonine (hCPA T268G); and b) a second agent comprising 3-cyclopentyl-alpha-phenylalanine- methotrexate (the pro-drug).3AFSDOCS:304317774.1VIA EFS Attorney Docket No. 044797.00005 Date of Deposit: December 22, 2025

[0012] A further aspect of the present disclosure is directed to a method of synthesizing the prodrug 3-cyclopentyl-α-phenylalanine-methotrexate.

[0013] By targeting tumor helper cells that infiltrate the tumor and aid in tumor growth, instead of targeting the tumor cells themselves, the presently disclosed methods and systems avoid having to target an unreliable, highly variable cancer antigen. TAMs are targetable infiltrating cells present in most aggressive tumors, and in virtually all aggressive, late-stage tumors. TAMs are hyperpolarized to the M2 phenotype, which facilitates cell-proliferation and tissue repair, and prevents inflammation and clearance of tumor cells. Higher densities of TAMs within the tumor microenvironment are associated with poor clinical outcomes. See, Wang et al., Front. Immunol.74:1264774 (2024).

[0014] By using an enzyme that differs from the endogenous human enzyme in terms of a single amino acid substitution and a pro-drug cleavable only by the mutated enzyme and not the endogenous wild-type enzyme, the problems of immune rejection and systematic pro-drug cleavage by endogenous enzymes may be overcome.

[0015] The working examples demonstrate that the pro-drug, 3-cyclopentyl-alpha- phenylalanine-methotrexate, is non-toxic and by the targeting to the cancer by means of the TAM specific anti-CD206 antibody, the disclosed methods and systems may effectively treat cancers characterized by solid tumors, such as glioblastomas.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIG. 1 schematically illustrates the components of the antibody directed enzyme pro-drug therapy (ADEPT) system and method of pro-drug activation.

[0017] FIG. 2 is an image of a western blot showing the presence of hCPA in the conditioned media (CM) and not in the cellular lysate (L), indicating that hCPA is produced and secreted in mammalian cells transfected and manufactured to express hCPA or mutant hCPA.

[0018] FIG. 3 is a set of images of western blots showing that the hCPA is isolated in the first three elutions of the nickel column as part of work to optimize protein purification, demonstrating that the His-tag attached to the wild-type or mutant hCPA can effectively be used to isolate and purify the expressed and secreted protein.

[0019] FIG. 4 is a set of images of western blots showing trypsin cleavage of the proenzyme into the active form of hCPA demonstrating effective production and activation in the system.4AFSDOCS:304317774.1VIA EFS Attorney Docket No. 044797.00005 Date of Deposit: December 22, 2025

[0020] FIG. 5 is an image of a western blot showing an optimal time for trypsin activation of hCPA as part of the optimization for enzyme purification and activation.

[0021] FIG. 6 is an image of a western blot showing activation of hCPA in 15 μg / mL trypsin for 1 hour as part of the optimization for enzyme purification and activation.

[0022] FIG. 7 is a set of immunohistochemistry images showing macrophages in rat glioblastoma that express CD206 (MMR) within the tumor microenvironment, but not in the surrounding normal brain tissue demonstrating the specificity of CD206 expression by TAMs within the tumor.

[0023] FIG. 8 is a set of immunohistochemistry images showing CD206+(MMR+) macrophages in human glioblastoma within the tumor microenvironment. The image on the left is stained with DAPI, anti-macrophages, and pre-immune IgG control for CD206, thus demonstrating the lack of background signal. The image in the middle is stained for anti-CD206 demonstrating strong signal in the malignant tumor sample, and the image on the right is stained the same but is from a section of normal brain (non-cancerous) tissue from the same individual demonstrating a lack of CD206+cells within the normal, non-cancerous brain tissue. This again demonstrates the specificity of CD206 expression by TAMs within the tumor microenvironment and not within the normal brain tissue from human patient tissue.

[0024] FIG. 9 is an immunohistochemistry image showing CD206+(MMR+) macrophages in a second human glioblastoma demonstrating consistency of CD206 presence in human tumors.

[0025] FIG. 10 schematically illustrates pro-drug 3-cyclopentyl-alpha-phenylalanine- methotrexate and its cleavage by mutant hCPA to active methotrexate.

[0026] FIG. 11 schematically illustrates the 3-D structure of the active site of human carboxypeptidase A wild-type (left) and T268G mutant (right).

[0027] FIG. 12 is a line graph of a toxicity study of methotrexate and the pro-drug 3-cyclopentyl- alpha-phenylalanine-methotrexate.

[0028] FIG. 13 is a schematic illustration of an embodiment of the inventive 7-step synthesis of the pro-drug 3-cyclopentyl-alpha-phenylalanine-methotrexate.DETAILED DESCRIPTION OF THE DISCLOSURE

[0029] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the subject matter herein5AFSDOCS:304317774.1VIA EFS Attorney Docket No. 044797.00005 Date of Deposit: December 22, 2025belongs. As used in the specification and the appended claims, unless specified to the contrary, the following terms have the meaning indicated to facilitate the understanding of the present disclosure.

[0030] As used in the description and the appended claims, the singular forms “a”, “an”, and “the” mean “one or more” and therefore include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a composition” includes mixtures of two or more such compositions, reference to “an inhibitor” includes mixtures of two or more such inhibitors, and the like.

[0031] Unless stated otherwise, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. “About” can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term “about.”

[0032] The term “approximately” as used herein refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0033] The transitional term “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. By contrast, the transitional phrase “consisting of’ excludes any element or method step not specified in the claim (or the specific element or method step with which the phrase “consisting of’ is associated). The transitional phrase “consisting essentially of’ limits the scope of a claim to the specified elements and method or steps and “unrecited elements and method steps that do not materially affect the basic and novel characteristic(s)” of the claimed disclosure.

[0034] The term “room temperature” as used herein refers to a temperature which is typically in the range of about 20 °C to about 25 °C.Antibody-enzyme conjugate

[0035] In one aspect, the present disclosure provides a method of treating a cancer characterized by the presence of a solid tumor. The method entails, administering to a human patient in need thereof a first agent comprising an antibody or fragment thereof that binds a CD206 receptor (also 6AFSDOCS:304317774.1VIA EFS Attorney Docket No. 044797.00005 Date of Deposit: December 22, 2025referred to as mannose macrophage receptor (MMR)) present on tumor associated macrophages (TAMS), conjugated to a mutant of human carboxypeptidase A (hCPA) wherein the mutant contains a glycine residue at position 268 in place of threonine (hCPA T268G); followed by administering to the patient a second agent comprising 3-cyclopentyl-alpha-phenylalanine- methotrexate.

[0036] An embodiment of the present ADEPT system and method is schematically illustrated in FIG. 1. It includes an anti-CD206 antibody that binds TAMs and not a tumor-specific marker. The enzyme, hCPA T268G cleaves 3-cyclopentyl-alpha-phenylalanine-methotrexate into the cytotoxic drug, methotrexate. The enzymatic cleavage is illustrated in FIG. 10.Anti-CD206 Antibody or fragment thereof

[0037] The antibody or fragment thereof binds CD206. CD206, also known as mannose macrophage receptor (MMR), mediates endocytosis of glycoproteins by macrophages. CD206 binds proteins and lipids conjugated with mannose on the surface of potentially pathogenic viruses, bacteria, and fungi for neutralization by phagocytic engulfment (Fiani et al., Int. J. Mol. Sci.21(17):6318 (2020) and Cummings, Curr. Opin. Struct. Biol. 75:102394 (2022)). CD206 is known as a marker for TAMs (Dong et al., Int. J. Mol. Sci. 17(3):320 pp. 1-12 (2016), Zarif et al., Eur. Urol. Oncol. 2(4):429-436 (2019), Fiani et al., Int. J. Mol. Sci. 21(17):6318 (2020), Yin et al., Adv. Sci. (Weinh). 9(9):2102620 p. 1-14 (2022), Guo et al., Curr. Cancer Drug Targets 22(11):904-918 (2022), and Albakri et al., J. Leukoc. Biol. 112(4):617-628 (2022)).

[0038] Anti-CD206 antibodies are known in the art. See, International Patent Application Publication No. WO 2022 / 244852, U. S. Patent 9,913,920, and Chinese Patent 114316060.

[0039] Amino acid sequences of representative anti-CD206 antibody variable heavy (VH) and variable light (VL) regions are set forth herein.

[0040] In some embodiments, the anti-CD206 VH region has the amino acid sequence set forth below (SEQ ID NO: 1):1 qvqlqqpgae Ivrpgssvkl sckasgytfi sywmdwvkqr pgqglewign ifpsdsethy 61 nqkfrdkatl tvdkssstas ihlssltsed savyycgrrd ssgwidywgq gttltvss

[0041] In some embodiments, the anti-CD206 VL region has the amino acid sequence set forth below (SEQ ID NO: 2):1 diqmtqttss Isaslgdrvt iscrasqdis nylnwyqqkp dgtvklliyy tsrfhsgvps 61 rfsgsgsgtd ysltihnveq ediatyfcqq ssmlpftfgs gtklemk7AFSDOCS:304317774.1VIA EFS Attorney Docket No. 044797.00005 Date of Deposit: December 22, 2025

[0042] In some embodiments, the anti-CD206 VH region has the amino acid sequence set forth below (SEQ ID NO: 3):1 qvqlqqpgae Ivkprasvkl scrasgyifi sywmqwvkqr pgqglewige idpsdsyiny 61 nqkfkgkasl tvdtssntay mqlssltsed savyycatgd mgddfyamdy wgqgtsvtvs 121 s

[0043] In some embodiments, the anti-CD206 VL region has the amino acid sequence set forth below (SEQ ID NO: 4):1 divmnqdels npvtsgesvs iscrssksll ykdgktylnw flqrpgqspq lliylmstra 61 sgvsdrfsgs gsgtdftlei srvkaedvgv yycqqlveyp ftfgggtkle ik

[0044] In some embodiments, the anti-CD206 VH region has the amino acid sequence set forth below (SEQ ID NO: 5):1 qvqlqqpgae Ivkpgasvkv sckasgytft tywmhwvkqr pgqglewigr ihpsdsdsny 61 nqkfkgkatl tvdkssstay mqlssltsed savyycaigr gfaywgqgtl vtvsa

[0045] In some embodiments, the anti-CD206 VL region has the amino acid sequence set forth below (SEQ ID NO: 6):1 divltqspas lavslgqraa iscrasesvd nygnsfmhwy qqkpgqppkl liyrasnles 61 giparfsgsg srtdftltin pveaddvaty ycqqgnedpy tfgggtklei k

[0046] In some embodiments, the anti-CD206 VH region has the amino acid sequence set forth below (SEQ ID NO: 7):1 qvqlqqsgae Ivrpgtsvkv sckasgyaft nyliewvkqr pgqglewigv inpgsggtny 61 nekfkgkatl tadkssstay mqlssltsed savyfcardy gstfdywgqg ttltvss

[0047] In some embodiments, the anti-CD206 VL region has the amino acid sequence set forth below (SEQ ID NO: 8):1 divltqspat Isvtpgdsvs Iscrasqsis snlhwyqqks hesprllinf asqsisgips 61 rfsgsgsgtd ftlnintvet edfgmyfcqq snswpltfga gtklelk

[0048] In some embodiments, the anti-CD206 VH region has the amino acid sequence set forth below (SEQ ID NO: 9):1 qvqlqqsgae Ivrpgtsvkv sckasgyaft dyliewvkqr pgqglewigv inpgrggtny 61 nekfkgkatl tadkssstay mqlssltsed savyfcaryy grsfgyfdvw gtgttvtvss

[0049] In some embodiments, the anti-CD206 VL region has the amino acid sequence set forth below (SEQ ID NO: 10):1 diqmtqspas qsaslgesvt itclasqtig twlawyqqkp gkspqlliya atsladgvps 61 rfsgsgsgtk fsfkisslqa edfvsyycqq lysppltfga gtklelk

[0050] In some embodiments, the anti-CD206 VH region has the amino acid sequence set forth below (SEQ ID NO: 11):1 qvqlqqpgae Ivrpgssvkl sckasgytft sywmdwvkqr pgqglewign iypsdsethy 61 nqkfkdratl tvdkssttay mqlssltsed savyyctrrd ssgwidywgq gttltvss8AFSDOCS:304317774.1VIA EFS Attorney Docket No. 044797.00005 Date of Deposit: December 22, 2025

[0051] In some embodiments, the anti-CD206 VL region has the amino acid sequence set forth below (SEQ ID NO: 12):1 diqmtqttss Isaslgdrvt iscrasqdis nylnwyqqkp dqtvklliyy tsrlhsqvps 61 rfsqrgsgtd ysltisnleq ediatyfcqq gntlpftfgs gtqleik

[0052] In some embodiments, the anti-CD206 VH region has the amino acid sequence set forth below (SEQ ID NO: 13):1 evqlvesggg Ivqpkgslkl scaasgfsfn tytmnwvrqa pgkglewvar irsksnnyet 61 yyadsvkdrf tisrddsesm lylqmnnlkt edtamyycvs pdsdswfayw gqgtlvtvsa

[0053] In some embodiments, the anti-CD206 VL region has the amino acid sequence set forth below (SEQ ID NO: 14):1 diqmnqspss Isaslgdtit iachasqnin vwlswyqqkp gnipklliyk asnlhtgvps 61 rfsgsgsgtg ftltisslqp ediatyycqq gqsypltfgg gtkleik

[0054] In some embodiments, the anti-CD206 VH region has the amino acid sequence set forth below (SEQ ID NO: 15):1 qvqlqqpgae Ivkpgasvkm sckasgytfs tywitwvkqr pgqglewigd iypggdstny 61 nekfkskatl tedtssstvy mqlssltseh savyycargi tggyfdywgq gttltvss

[0055] In some embodiments, the anti-CD206 VL region has the amino acid sequence set forth below (SEQ ID NO: 16):1 diqmtqttss Isaslgdrvt itcrasqdis nylnwyqrkp dgtvrlliyy tsglhsgvps 61 rfsgsgsgtd ysltisnleq edfatyfcqq ghtlpwtfgg gtnleik

[0056] In some embodiments, the anti-CD206 VH region has the amino acid sequence set forth below (SEQ ID NO: 17):1 qvqlqqpgae Ivrpgssvkl sckasgytft tywmhwvkqr piqglewign idpsdsethy 61 nqkfkdkatl tvdkssstay mqlnsltsed savyycargd gyyvyfaywg qgttltvss

[0057] In some embodiments, the anti-CD206 VL region has the amino acid sequence set forth below (SEQ ID NO: 18):1 eivltqspal maaspgekvt itcsvsssis ssnlhwyqqk setspkpwiy atsnlasgvp 61 vrfsgsgsgt sysltissma aedaatyfcq qwssfpltfg agtklelk

[0058] In some embodiments, the anti-CD206 VH region has the amino acid sequence set forth below (SEQ ID NO: 19):1 qvqllqsgae Ivrpgtavkm sckaseytft nywigwarqr pghglewigd iypgggyiny 61 nekfkgkatl tadkssstvy mqfssltsed saiyycargg snyvghfdyw gqgttltvss

[0059] In some embodiments, the anti-CD206 VL region has the amino acid sequence set forth below (SEQ ID NO: 20):1 diqmtqttss Isaslgdrvt iscrasqdis nylnwyqqkp dgtvklliyy tsilhsrvps 61 rfsgsgsgtd ysltisnleq ediatyfcqh ghtlpwtfgg gtkleik

[0060] In some embodiments, the anti-CD206 VH region has the amino acid sequence set forth below (SEQ ID NO: 21):9AFSDOCS:304317774.1VIA EFS Attorney Docket No. 044797.00005 Date of Deposit: December 22, 20251 evqlvesggg Ivgpggslrl scaasgfnik cspgakwvrg apgkglewva cspgakrfti 61 sadtskntay lqmnslraed tavyycsrcs pgakwgqgtl vtvss

[0061] In some embodiments, the anti-CD206 VL region has the amino acid sequence set forth below (SEQ ID NO: 22):1 diqmtqspss Isasvgdrvt itcrasqdvn tavawyqqkp gkapklliys asflysgvps 61 rfsgsrsgtd ftltisslqp edfatyycqq hyttpptfgq gtkveik

[0062] In some embodiments, the anti-CD206 antibody is an immunoglobulin single variable domain (VHH). In some embodiments, the anti-CD206 VHH has the amino acid sequence set forth below (SEQ ID NO: 23):1 qvqlqesggg Ivqagdslrl scaasgrtfs inymgwyrqa pgkqrelvaa itsgsgstny 61 adsvkgrfti srdnakktmy Iqmnslkped tavyycnadm dsslsggyvd vwgqgtqvtv 121 ss

[0063] In some embodiments, the anti-CD206 VHH has the amino acid sequence set forth below (SEQ ID NO: 24):1 qvqlqesggg Ivqaggslrl scaasgftdd dydigwfrqa pgkeregvsc isssdgstyy 61 adsvkgrfti ssdnakntvy Iqmnslkped tavyycaadf frwdsgsyyv rgcrhatydy 121 wgqgtqvtvs s

[0064] In some embodiments, the anti-CD206 antibody or fragment thereof is clone C068C2 (BioLegend Inc.), clone 19.2 (BD Pharmingen), clone MR5D3 (Bio Rad), clone 15-2 (Abeam and LSBio), clone 685645 (R& D Systems).

[0065] The anti-CD206 antibody or fragment thereof is not limited to known antibodies. Methods of generating new antibodies against a specific antigen are well known in the art. For example, a course of antigen (e., CD206) is administered to an animal, e.g., a mouse, a rabbit, or a camelid, over time. After administration of the antigen, blood samples are taken from the animal, antibodies are isolated, and the isolated antibodies are tested for their ability to bind the antigen. In addition, cell lines expressing a specific anti-CD206 antibody, including B-cell lines (hybridomas) can be isolated and propagated to collect antibodies from tissue culture. Antibody characterization and optimization, e.g., complementarity-determining region (CDR) mutation, CDR grafting, or CDR shuffling are well known in the art. See, U. S. Patent 5,225,539 5,530,101, 5,585,089, 5,693,762, 6,180,370, 6,399,763, 6,838,254, 7,214,775, 8,388,968 8,580,927, and 8,778,345, U. S. Patent Application Publication No. 2007 / 0298443, and Jones et al., Nature 321(6069):522-525 (1986), Riechmann et al., Nature 332 (6162):323 -7 (1988), and Queen et al., Proc. Natl. Acad. Sci. U. S. A. 86(24)-.10029-33 (1989).

[0066] Making (e.g., deriving) antibody fragments (e.g., a single-chain antibody fragment (scFv)) from antibodies is known in the art. The term “derived from” as used herein when referring 10AFSDOCS:304317774.1VIA EFS Attorney Docket No. 044797.00005 Date of Deposit: December 22, 2025to a protein and nucleic acid refers to a sequence that originates and is identified from the sequence of a parent (e.g., wild-type or endogenous) protein and nucleic acid, respectively. A sequence derived from a parent sequence may be a portion (fragment) of the parent sequence and / or may vary from the parent amino acid or nucleotide sequence by at least one position. Protein variants may include amino acid substitutions, insertions, and / or deletions. For example, an amino acid sequence derived from a parent sequence may constitute a fragment of the parent sequence and be identical for a specific range of amino acids of the parent but does not include amino acids outside that specific region. Nucleic acid variants may include substitutions or in-frame insertions or deletions (z.e., insertions or deletions that do not result in downstream frame shift of the nucleic acid codons). For example, anti-CD206 scFv antibody fragments may be easily prepared based on combinations of VH and VL sequences described above, joined together using linkers commonly used for this purpose.

[0067] In some embodiments, the linker has the amino acid GGGX, GGGGX (SEQ ID NO: 25), or GSSGSX (SEQ ID NO: 26), where X is any nucleotide, typically either cysteine (C) or serine (S), or repeating sequence thereof. In some embodiments, the linker has the amino acid sequence GGGGS (SEQ ID NO: 27), GSPRG (SEQ ID NO: 28), GGGGSGGGGS (SEQ ID NO: 29), GGGGSGGGGSGGGGS (SEQ ID NO: 30), GGGGS GGGGS GGGGS GGGGS (SEQ ID NO: 31), GSPRGGGGSGGGGSGGGGS (SEQ ID NO: 32), GSTSGSGKPGSGEGSTKG (SEQ ID NO: 33), KESGSVSSEQLAQFRSLD (SEQ ID NO: 34), EGKSSGSGSESKST (SEQ ID NO: 35), or GSAGSAAGSGEF (SEQ ID NO: 36).Enzyme - mutant human carboxypeptidase A (hCPA)

[0068] The antibody (or fragment thereof) is conjugated with a mutated hCPA, which contains a glycine amino acid residue at position 268 in place of threonine (T268G). The methotrexate (MTX) pro-drug 3-cyclopentyl-alpha-phenylalanine-methotrexate contains a bulky steric group preventing it from being cleaved by endogenous, wildtype hCPA. Threonine at amino acid position 268 is located in the hCPA active site, making large, aromatic C-terminus residues unable to bind the enzyme (FIG. 11, left panel). However, the non-endogenous mutant hCPA T268G is capable of enzymatically cleaving the modified pro-drug MTX. The presence of glycine in lieu of threonine at position 268 opens the active site of hCPA, allowing 3-cyclopentyl-alpha-phenylalanine-11AFSDOCS:304317774.1VIA EFS Attorney Docket No. 044797.00005 Date of Deposit: December 22, 2025methotrexate to enter the active site and be cleaved (FIG. 11, right panel and FIG. 10). See, Smith etal., J. Biol. Chem. 72(25)-.15804-16 (1997).

[0069] The amino acid sequences of representative immature hCPA(T268G) are based on NCBI Accession Nos. NP_001859 and NP_002429.1, both incorporated herein by reference. The amino acid sequence of a representative mature and active hCPA(T268G) is set forth below (SEQ ID NO: 37):1 mrgllvlsvl Igavfgkedf vghqvlrisv adeaqvqkvk eledlehlql dfwrgpahpg 61 spidvrvpfp siqavkifle shgisyetmi edvqslldee qeqmfafrsr arstdtfnya 121 tyhtleeiyd fldllvaenp hlvskiqign tyegrpiyvl kfstggskrp aiwidtgihs 181 rewvtqasgv wfakkitqdy gqdaaftail dtldifleiv tnpdgfafth stnrmwrktr 241 shtagslcig vdpnrnwdag fglsgassnp csetyhgkfa nsevevksiv dfvkdhgnik 301 afisihsysq llmypygykt epvpdqdeld qlskaavtal aslygtkfny gsiikaiyqa 361 sgstidwtys qgikysfgfe Irdtgrygfl Ipasqiipta ketwlallti mehtlnhpy

[0070] In the above sequence, T268 corresponds to T378 (position 378, with a mutant glycine boxed in SEQ ID NO: 37) in the immature hCPA amino acid sequence, which includes a 110 amino acid long pre-pro sequence. In some embodiments, the hCPA is produced as a pre-pro hCPA, in which the hCPA contains a “pre” region containing a signal sequence of amino acids 1 to about 25 that targets the protein for insertion into the endoplasmic reticulum for subsequent secretion and a “pro” region of amino acids about 27 to about 110 that prevents premature activation of the enzyme in vivo. Inclusion of the full pre-pro-hCPA may allow for secretion in mammalian cell culture and subsequent activation, either before or after linking hCPA with the antibody.

[0071] The nucleic acid sequences encoding a representative hCPA(T268G) may be prepared based on nucleic acid sequences encoding native or wild-type hCPA, as described for example, in NCBI Accession Nos. NM_001868.2 and NM_002438.4, both incorporated herein by reference. The nucleic acid sequence of a representative wildtype hCPA is set forth below (SEQ ID NO: 38):1 cagctgggca gctctgggaa cttggattag gtggagaggc agttgggggg cctcgttgtt 61 ttgcgtctta gttccgccct cctgtccatc aggagaagga aaggataaac cctgggccat 121 gaggctaccc ctgctcctgg tttttgcctc tgtcattccg ggtgctgttc tcctactgga 181 caccaggcaa tttttaatct ataatgaaga tcacaagcgc tgcgtggatg cagtgagtcc 241 cagtgccgtc caaaccgcag cttgcaacca ggatgccgaa tcacagaaat tccgatgggt 301 gtccgaatct cagattatga gtgttgcatt taaattatgc ctgggagtgc catcaaaaac 361 ggactgggtt gctatcactc tctatgcctg tgactcaaaa agtgaatttc agaaatggga 421 gtgcaaaaat gacacacttt tggggatcaa aggagaagat ttatttttta actacggcaa 481 cagacaagaa aagaatatta tgctctacaa gggatcgggt ttatggagca ggtggaagat 541 ctatggaacc acagacaatc tgtgctccag aggttatgaa gccatgtata cgctactagg 601 caatgccaat ggagcaacct gtgcattccc gttcaagttt gaaaacaagt ggtacgcaga 661 ttgcacgagt gctgggcggt cggatggatg gctctggtgc ggaaccacta ctgactatga 721 cacagacaag ctatttggat attgtccatt gaaatttgag ggcagtgaaa gcttatggaaAFSDOCS:304317774.1VIA EFS Attorney Docket No. 044797.00005 Date of Deposit: December 22, 2025781 taaagacccg ctgaccagcg tttcctacca gataaactcc aaatccgctt taacgtggca 841 ccaggcgagg aaaagctgcc aacaacagaa cgctgagctc ctgagcatca cagagattca 901 tgagcaaaca tacctgacag gattaaccag ttccttgacc tcaggactct ggattggact 961 taacagtctg agcttcaaca gcggttggca gtggagtgac cgcagtcctt tccgatattt 1021 gaactggtta ccaggaagtc catcagctga acctggaaaa agctgtgtgt cactaaatcc 1081 tggaaaaaat gctaaatggg aaaatctgga atgtgttcag aaactgggct atatttgcaa 1141 aaagggcaac accactttaa attcttttgt tattccctca gaaagtgatg tgcctactca 1201 ctgtcctagt cagtggtggc cgtatgccgg tcactgttac aagattcaca gagatgagaa 1261 aaaaatccag agggatgctc tgaccacctg caggaaggaa ggcggtgacc tcacaagtat 1321 ccacaccatc gaggaattgg actttattat ctcccagcta ggatatgagc caaatgacga 1381 attgtggatc ggcttaaatg acattaagat tcaaatgtac tttgagtgga gtgatgggac 1441 ccctgtaacg tttaccaaat ggcttcgtgg agaaccaagc catgaaaaca acagacagga 1501 ggattgtgtg gtgatgaaag gcaaggatgg gtactgggca gatcggggct gtgagtggcc 1561 tcttggctac atctgcaaga tgaaatcacg aagccaaggt ccagaaatag tggaagtcga 1621 aaaaggctgc aggaaaggct ggaaaaaaca tcacttttac tgctatatga ttggacatac 1681 gctttcaaca tttgcagaag caaaccaaac ctgtaataat gagaatgctt atttaacaac 1741 tattgaagac agatatgaac aagccttcct gactagtttc gttggcttaa ggcctgaaaa 1801 atatttctgg acaggacttt cagatataca aaccaaaggg acttttcagt ggaccatcga 1861 ggaagaggtt cggttcaccc actggaattc agatatgcca gggcgaaagc cagggtgtgt 1921 tgccatgaga accgggattg cagggggctt atgggatgtt ttgaaatgtg atgaaaaggc 1981 aaaatttgtg tgcaagcact gggcagaagg agtaacccac ccaccgaagc ccacgacgac 2041 tcccgaaccc aaatgtccgg aggattgggg cgccagcagt agaacaagct tgtgtttcaa 2101 gctgtatgca aaaggaaaac atgagaagaa aacgtggttt gaatctcgag atttttgtcg 2161 agctctgggt ggagacttag ctagcatcaa taacaaagag gaacagcaaa caatatggcg 2221 attaataaca gctagtggaa gctaccacaa actgttttgg ttgggattga catatggaag 2281 cccttcagaa ggttttactt ggagtgatgg ttctcctgtt tcatatgaaa actgggctta 2341 tggagaacct aataattatc aaaatgttga atactgtggt gagctgaaag gtgaccctac 2401 tatgtcttgg aatgatatta attgtgaaca ccttaacaac tggatttgcc agatacaaaa 2461 aggacaaaca ccaaaacctg agccaacacc agctcctcaa gacaatccac cagttactga 2521 agatgggtgg gttatttaca aagactacca gtattatttc agcaaagaga aggaaaccat 2581 ggacaatgcg cgagcgtttt gcaagaggaa ttttggtgat cttgtttcta ttcaaagtga 2641 aagtgaaaag aagtttctat ggaaatatgt aaacagaaat gatgcacagt ctgcatattt 2701 tattggttta ttgatcagct tggataaaaa gtttgcttgg atggatggaa gcaaagtgga 2761 ttacgtgtct tgggccacag gtgaacccaa ttttgcaaat gaagatgaaa actgtgtgac 2821 catgtattca aattcagggt tttggaatga cattaactgt ggctatccaa acgccttcat 2881 ttgccagcga cataacagta gtatcaatgc taccacagtt atgcctacca tgccctcggt 2941 cccatcaggg tgcaaggaag gttggaattt ctacagcaac aagtgtttca aaatctttgg 3001 atttatggaa gaagaaagaa aaaattggca agaggcacga aaagcttgta taggctttgg 3061 agggaatctg gtctccatac aaaatgaaaa agagcaagca tttcttacct atcacatgaa 3121 ggactccact ttcagtgcct ggactgggct gaatgatgtc aattcagaac acacgttcct 3181 ttggacggat ggacgaggag tccattacac aaactggggg aaaggttacc ctggtggaag 3241 aagaagcagt ctttcttatg aagatgctga ctgtgttgtt attattggag gtgcatcaaa 3301 tgaagcagga aaatggatgg atgatacctg cgacagtaaa cgaggctaca tatgccagac 3361 acgatccgac ccttccttga ctaatcctcc agcaacgatt caaacagatg gctttgttaa 3421 atatggcaaa agcagctatt cactcatgag acaaaaattt caatggcatg aagcggagac 3481 atactgcaag cttcacaatt cccttatagc cagcattctg gatccctaca gtaatgcatt 3541 tgcgtggctg cagatggaaa catctaatga acgtgtgtgg atcgccctga acagtaactt 3601 gactgataat caatacactt ggactgataa gtggagggtg aggtacacta actgggctgc 3661 tgatgagccc aaattgaaat cagcatgtgt ttatctggat cttgatggct actggaagac 3721 agcacattgc aatgaaagtt tttactttct ctgtaaaaga tcagatgaaa tccctgctac 3781 tgaaccccca caactgcctg gcagatgccc ggagtcagat cacacagcat ggattcctttAFSDOCS:304317774.1VIA EFS Attorney Docket No. 044797.00005 Date of Deposit: December 22, 20253841 ccatggtcac tgttactata ttgagtcctc atatacaaga aactggggcc aagcttctct 3901 ggaatgtctt cgaatgggtt cctctctggt ttccattgaa agtgctgcag aatccagttt 3961 tctgtcatat cgggttgagc cacttaaaag taaaaccaat ttttggatag gattgttcag 4021 aaatgttgaa gggacgtggc tgtggataaa taacagtccg gtctcctttg tcaactggaa 4081 cacaggagat ccctctggtg aacggaatga ttgtgtagct ttacatgcgt cttctgggtt 4141 ttggagtaat attcactgtt catcctacaa aggatatatt tgtaaaagac caaaaattat 4201 tgatgctaaa cctactcatg aattacttac aacaaaagct gacacaagga agatggaccc 4261 ttctaaaccg tcttccaacg tggccggagt agtcatcatt gtgatcctcc tgattttaac 4321 gggtgctggc cttgccgcct atttctttta taagaaaaga cgtgtgcacc tacctcaaga 4381 gggcgccttt gaaaacactc tgtattttaa cagtcagtca agcccaggaa ctagtgatat 4441 gaaagatctc gtgggcaata ttgaacagaa tgaacactcg gtcatctagt acctcaatgc 4501 gattctgaga tatttgaatt tcataaaatt gtaactgaaa tttaaaattt ttagttcaat 4561 gtgattgttt tctttaaaat gagtactgaa ttgtactggt ctgtcctttt ttcctttgcc 4621 taattgaaga aataattgct tgttttctag cctggcaaga tattttcata aaagagggat 4681 aacaatgctg attactacct tttaaaatat tttagataaa tgcacagcac cacagcacca 4741 catctaagca ttagtgatgg gtagctgatg tcagcttcat gtggatttta agcactctag 4801 aaacaatgaa gcttcttggc atattttaag gagctcccaa aatgtgttac ctattaaatt 4861 gtaactcagc aagtagaaga ccatttgaaa agtcaggtac aaatttcctc aagtggcata 4921 aaaatgtagt cagttttctc ttttaccagt ttttatttcc actccaatta tttagaactt 4981 tatttgtaca tgtgcagaag aataaggcag ctgagaatct tgtttccccc aagagagttt 5041 tacaggctga gtgttgcaaa tgtgttcttt gtcctgttat atgtatatca ggaatacaag 5101 gatgtgaaat aaaactgtaa atttgcataa ctggatgtac ttagataatg tgaaataaac 5161 attaaagaca aggtctattt ttaataga

[0072] In some embodiments, mutant T268G hCPA is synthesized using site-directed mutagenesis of an appropriate wildtype hCPA (e.g., a hCPA-encoding plasmid) and altering the nucleotides to convert the threonine-encoding codon (e.g., ACC) correlating to amino acid position 268 to a glycine-encoding codon (e.g. GGC). Representative glycine-encoding codons include GGA, GGC, GGT and GGG. In some embodiments, the glycine-encoding codon is GGC.Conjugation of mutant enzyme to antibody or fragment thereof

[0073] Methods of conjugating (connecting) the anti-CD206 antibody or binding fragment thereof and to the hCPA T268G may be conducted in accordance with standard techniques. In some embodiments they may be connected covalently. In some other embodiments, they are expressed as a fusion protein.

[0074] Covalent connections may be established with techniques known in the art, with for example, use of homobifunctional, heterobifunctional, and photoreactive crosslinking reagents. Representative homobifunctional crosslinking reagents include amine-to-amine crosslinking reagents and sulfhydryl-to-sulfhydryl crosslinking reagents. Representative amine-to-amine crosslinkers includedisuccinimidyl glutarate (DSG), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl)suberate (BS3), Bis-succinimide ester polyethylene glycol (PEG) 400AFSDOCS:304317774.1VIA EFS Attorney Docket No. 044797.00005 Date of Deposit: December 22, 2025(BS(PEG)9), dithiobis(succinimidyl propionate) (DSP), 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP), ethylene glycol bis(succinimidyl succinate) (EGS), and sulfo-EGS. Representative sulfhydryl-to-sulfhydryl crosslinkers include succinimidyl 4-(N-maleimidomethyl) cyclohexane- 1 -carboxylate (SMCC).

[0075] Representative heterobifunctional crosslinking reagents include amine-to-sulfhydryl, amine-to-azine crosslinking reagents, amine-to-carboxyl crosslinking reagents, amine-to-azine crosslinking reagents, carbohydrate-to-sulfhydryl crosslinking reagents.

[0076] Representative amine-to-sulfhydryl crosslinking reagents include sulfo-SMCC, succinimidyl 3-(2-pyridyldithio)propionate (SPDP), succinimidyl 6-(SPDP)hexanoate (LC- SPDP), and Sulfo-LC-SPDP. Representative amine-to-azine crosslinking reagents include tetrafluorophenyl (TFP) ester-PEG4-dibenzylcyclooctyne (DBCO), TFP ester-PEG12-DBCO, NHS-phosphine, l-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), NHS-azide, andNHS- PEG-azide. Representative amine-to-carboxyl crosslinkers include l-ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), sulfo- NHS. Representative carbohydrate-to-sulfhydryl crosslinking reagents include 4-(4-N- maleimidophenyl)butyric acid hydrazide (MPBH), N-s-maleimidocaproic acid hydrazide (EMCH), N-P-maleimidopropionic acid hydrazide (BMPH), 3-(2-pyridyldithio)propionyl hydrazide (PDPH), and N-K-maleimidoundecanoic acid hydrazide (KMUH).

[0077] Representative photoreactive crosslinkers include sulfosuccinimidyl 6-(4'-azido-2'- nitrophenylamino)hexanoate (sulfo-SANPAH), succinimidyl 4,4'-azipentanoate (SDA), sulfo- SDA, succinimidyl 6-(4,4'-azipentanamido)hexanoate (LC-SDA), sulfo-LA-SDA, succinimidyl 2- ((4,4'-azipentanamido)ethyl)-l,3'-dithiopropionate (SDAD), and sulfo-SDAD.

[0078] Additional conjugation methods that may be useful are known in the art, see, e.g., U. S.Patents 4,975,278, 6,051,576, 7,375,234, 7,498,298, 7,763,736, 8,101,238, 8,372,986, 8,394,914, 8,877,170, 8,927,682, 8,927,736, 9,302,997, 9,896,547, 9,988,408, 10,234,415, 10,501,412, 10,836,796, and 11,021,737, 11,028,185, 11,091,588, and 11,352,460, U. S. Patent Application Publication Nos. 2009 / 0069561, 2017 / 0183414, 2019 / 0241938, 2020 / 0239946, 2022 / 0118102, and O'Sullivan and Marks, Methods Enzymol. 73(Pt B)'.147-66 (1981).

[0079] In some embodiments, the antibody-enzyme conjugate is provided in the form of a fusion protein. Antibody-enzyme conjugate fusion proteins contain an antibody or fragment thereof that binds CD206 connected by a peptide bond to the enzyme or a fragment thereof that contains the 15AFSDOCS:304317774.1VIA EFS Attorney Docket No. 044797.00005 Date of Deposit: December 22, 2025enzymatic activity to cleave the pro-drug. Antibody-enzyme fusion proteins may be constructed using recombinant DNA techniques well known in the art. See, e.g., U. S. Patents 4,975,278, 5,314,995, 6,132,722, and 7,091,321, U. S. Patent Application Publication No. 2024 / 0190978, and Neuberger et al., Nature 312(5995):604-8 (1984).Pro-drug - 3-cyclopentyl-alpha-phenylalanine-methotrexate

[0080] The structure of 3-cyclopentyl-alpha-phenylalanine-methotrexate is as follows:

[00081] H2N N (3-cyclopentyl-alpha- phenylalanine-methotrexate).

[0082] The pro-drug and synthesis thereof are known in the art, See, e.g., Smith et al., J. Biol. Chem. 272(25 / 15804-16 (1997) and U. S. Patent 6,140,100. The pro-drug 3-cyclopentyl-alpha- phenylalanine-methotrexate is non-toxic to human cells (FIG. 10). It is cleaved by T268G hCPA, into the active methotrexate and 3-cyclopentyl-phenylalanine. A further aspect of the disclosure directed to a novel synthesis of the prodrug, is set forth below, and an embodiment thereof is presented in a working example.Pharmaceutical compositions containing an antibody-enzyme conjugate or pro-drug

[0083] For purposes of practicing the disclosed methods, the antibody-enzyme conjugate / fusion protein and the pro-drug may be formulated into pharmaceutical compositions that may also include a pharmaceutically acceptable carrier and optionally a pharmaceutically acceptable excipient.

[0084] Compositions for administration are typically provided as sterile solid or liquid preparations. Representative examples of solid carriers include lactose (anhydrous), magnesium stearate, starch (e.g., pregelatinized starch and sodium starch glycolate), and sodium hydroxide pellets.AFSDOCS:304317774.1VIA EFS Attorney Docket No. 044797.00005 Date of Deposit: December 22, 2025

[0085] Solid preparations may be reconstituted and diluted into a liquid preparation before use, e.g., with carriers containing isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous solutions, which may be buffered to a selected pH. Liquid carriers include aqueous and non-aqueous carriers alike. Representative examples of liquid carriers include sterile water for injection, saline, Lactated Ringer Injection solution, phosphate buffered saline, soluble sugars e.g., dextrose, trehalose dihydrate, and sucrose), dimethyl sulfoxide (DMSO), ethanol, and suitable mixtures thereof. In some embodiments, the liquid carrier includes a protein dissolved or dispersed therein, representative examples include serum albumin (e.g., human serum albumin, recombinant human albumin), gelatin, and casein. In some embodiments, the liquid carrier includes a water- miscible polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like). Representative examples of excipients include citric acid, sodium chloride, sodium succinate, sugars, polyalcohols, and isotonic electrolyte solutions (.g., Plasma-Lyte®) to achieve the desired isotonicity (e.g., compatible with blood). Depending on the carrier, other excipients may be added, e.g., wetting, dispersing, or emulsifying agents, gelling and viscosity enhancing agents, preservatives, surfactants (e.g., a polysorbate) and the like as known in the art. In some embodiments, a composition containing an antibody-enzyme conjugate / fusion protein includes trehalose dihydrate, sodium citrate dihydrate citric acid monohydrate, and polysorbate 20 or polysorbate 80 diluted in sterile water for injection with a pH of about 6.6.Methods of using ADEPT to treat cancer

[0086] Another aspect of the present disclosure is directed to a method of treating cancer characterized by the presence of a solid tumor, comprising administering to a human patient in need thereof a first agent comprising an antibody or fragment thereof that binds a CD206 receptor (mannose macrophage receptor; MMR) present on tumor associated macrophages (TAMs), conjugated to a mutant of human carboxypeptidase A (hCPA) wherein the mutant contains a glycine residue at position 268 in place of threonine (hCPA T268G); followed by administering to the patient a second agent comprising 3-cyclopentyl-alpha-phenylalanine-methotrexate.

[0087] Cancers treatable in accordance with the disclosed antibody-enzyme conjugates and prodrugs broadly include cancers characterized by the presence of a solid tumor. The tumor microenvironments of these cancers contain relatively high numbers of TAMs having CD206 on their surface.17AFSDOCS:304317774.1VIA EFS Attorney Docket No. 044797.00005 Date of Deposit: December 22, 2025

[0088] The term “subject” (or “patient”) as used herein includes all members of the animal kingdom prone (or disposed) to or suffering from the indicated cancer. In some embodiments, the subject is a human. Therefore, a subject “having cancer” or “in need of’ treatment according to the present disclosure broadly embraces subjects who have been positively diagnosed, including subjects having active disease who may have been previously treated with one or more rounds of therapy, and subjects who are not currently being treated (e.g, in remission) but who might still be at risk of relapse, and subjects who have not been positively diagnosed but who are predisposed to cancer (e.g., on account of the basis of prior medical history and / or family medical history, or who otherwise present with a one or more risk factors such that a medical professional might reasonably suspect that the subject was predisposed to cancer).

[0089] The terms “treat”, “treating”, and “treatment” as used herein refer to the art-recognized indicia of therapeutic efficacy, intervention, process performed on, or the administration of an active agent to the subject in need thereof with the therapeutic objective (“therapeutic effect”) which may include any one or more of reversing, alleviating, ameliorating, inhibiting, diminishing, slowing down, arresting, stabilizing, or preventing or prolongation of the onset, progression, development, metastases, severity or recurrence of a symptom, improvement in survival time, total / complete or partial remission, complication or condition, or biochemical indicia associated with cancer. Remission may be indicated by no detectable cancer cells, fewer tumor cells, and / or tumor shrinkage.

[0090] In some embodiments, the subject is an adult subject. In some embodiments, the subject is a pediatric subject.

[0091] In some embodiments, the cancer is brain cancer (e.g., astrocytoma and gliomas such as glioblastomas), colon or rectal cancer (e.g., colorectal carcinoma (CRC), colon adenocarcinoma (COAD), gastric cancer, head and neck cancer (e.g., squamous cell carcinoma of the head and neck (SCCHN) also called head and neck squamous cell carcinoma (HNSC)), liver cancer (e.g., hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (IHC), angiosarcoma, hepatoblastoma) or prostate cancer (e.g., prostate adenocarcinoma (PRAD)), and stomach cancer (e.g., stomach adenocarcinoma (STAD)). Publications describing TAMs known to be present in the tumor microenvironment of various cancers are listed in Table 1.Table 1Cancer Type Publication describing TAMs in the tumor18AFSDOCS:304317774.1VIA EFS Attorney Docket No. 044797.00005 Date of Deposit: December 22, 2025Colorectal Yin etal., Adv. Sci. (Weinh). 9(9):2102620 p. 1-14 (2022)Gastric Guo etal., Curr. Cancer Drug Targets 22(17):904-918 (2022)Head and Neck Albakri etal., J. Leukoc. Biol. 112(4):617 -628 (2022) Hepatocellular Ding etal., PLoS One 14(10), e0223971 (2019)Dong etal., Int. J. Mol. Sci. 17(3):320 pp. 1-12 (2016)Prostate Zarif et al., Eur. Urol. Oncol. 2(4):429-436 (2019)00092] In some embodiments, the cancer is metastatic. In some embodiments, the cancer is non- metastatic (e.g., glioblastomas). In some embodiments, the method is performed on a glioblastoma patient following surgical resection.

[0093] In some embodiments, the compositions may be administered to a subject as monotherapy as a “front / first-line therapy”, i.e., as an initial treatment in subjects who have undergone no prior anti-cancer treatment, either alone or in combination with other treatments; or a “second-line,” follow-on therapy, as a treatment in subjects who have undergone a prior anti-cancer treatment regimen, either alone or in combination with other treatments; or as a “third-line,” “forth-line,” etc. treatments, either alone or in combination with other treatments. The compositions may be administered to subjects who have had previous treatments which were unsuccessful or partially successful but who became intolerant to a particular treatment, or as an adjuvant treatment, i.e., to prevent reoccurrence of cancer in a patient with no currently detectable disease or after surgical removal of a tumor.Administration of the Antibody-enzyme conjugate

[0094] The antibody-enzyme conjugate is administered to a subject in need thereof in an effective amount, which, as used herein, refers to an amount of antibody-enzyme conjugate to provide the desired effects, e.g., binding CD206 and providing enough enzyme to cleave the amount of the MTX pro-drug.

[0095] In some embodiments, the antibody-enzyme conjugate is administered parenterally, e.g., intravenously, intraperitonially, intratumorally, or subcutaneously.

[0096] In some embodiments, the dosage amounts of the antibody-enzyme conjugate may range from about 0.1 to about 20 mg of patient body weight (mg / kg). In some embodiments, the dosage (effective amount) of the antibody-enzyme conjugate is about 0.1 mg / kg, 0.25 mg / kg, about 0.5 mg / kg, 0.75 mg / kg, 1 mg / kg, about 1.5 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, or about 20 mg / kg.19AFSDOCS:304317774.1VIA EFS Attorney Docket No. 044797.00005 Date of Deposit: December 22, 2025

[0097] In some embodiments, the dosage amounts range from about 1 to about 125 mg per body surface area (U / m2). In some embodiments, the dosage (effective amount) of the antibody-enzyme conjugate is about 1 mg / m2, about 2 mg / m2, about 3 mg / m2, about 4 mg / m2, about 5 mg / m2, about 7 mg / m2, about 10 mg / m2, about 12 mg / m2, about 15 mg / m2, about 18 mg / m2, about 20 mg / m2, about 25 mg / m2, about 30 mg / m2, about 40 mg / m2, about 50 mg / m2, about 75 mg / m2, about 100 mg / m2, or about 125 mg / m2.

[0098] In some embodiments, the dosage amounts of antibody-enzyme conjugate is measured by enzyme activity units. In some embodiments, the dosage amounts range from about 500 to about 10,000 enzyme units (U) per body surface area. In some embodiments, the dosage (effective amount) of the antibody-enzyme conjugate is about 500 U / m2, about 750 U / m2, about 1,000 U / m2, about 1,250 U / m2, about 1,500 U / m2, about 2,000 U / m2, about 2,250 U / m2, about 2,500 U / m2, about 2,750 U / m2, about 3,000 U / m2, about 3,250 U / m2, about 3,500 U / m2, about 4,000 U / m2, about 4,500 U / m2, about 5,000 U / m2, about 5,500 U / m2, about 6,000 U / m2, about 7,000 U / m2, about 8,000 U / m2, about 9,000 U / m2, or about 10,000 U / m2.

[0099] Dosage based on body surface area and dosage based on weight may be used interchangeably and methods of conversion between the two units are known in the art.[000100] In some embodiments, the dosage amount of antibody-enzyme conjugate is selected to achieve a concentration of about 0.05 to about 1 pM at the tumor site. The dosage amount of the antibody-enzyme conjugate required to obtain a concentration at the tumor site may be determined in accordance with standard techniques, such as affinity studies involving the pro-drug and the hCPA T268G enzyme. See, e.g., Smith et al., J. Biol. Chem. 272(25 / 15804-16 (1997).[000101] In some embodiments, the antibody-enzyme conjugate is administered in a bolus. In some embodiments, the antibody-enzyme conjugate is administered as three bolus doses, with about an hour between each dose, into a fast-running intravenous infusion of 5% dextrose.[000102] In some embodiments, the antibody-enzyme conjugate is administered as an intravenous infusion over a period of time that generally ranges from about 15 to about 120 minutes. Representative infusion times are 30 minutes, 60 minutes, 90 minutes, and 120 minutes. In some embodiments, the infusion time is between 30 and 60 minutes. In some embodiments, the first administration is infused into a patient for 90 minutes and subsequent administrations are infused into a patient for 30 minutes. Additional antibody-enzyme conjugate doses and administration methods that may be useful are known in the art, see, e.g., U. S. Patents 4,975,278, 5,405,990,20AFSDOCS:304317774.1VIA EFS Attorney Docket No. 044797.00005 Date of Deposit: December 22, 20255,587,161, 6,903,203, and 8,591,900, International Patent Application Publications WO 88 / 07378 and WO 89 / 10140, and Francis et al., Br. J. Cancer 87(6):600-7 (2002).Pro-drug Administration[000103] The methotrexate pro-drug is administered to a subject in need thereof in an effective amount, which as used herein, refers to an amount of pro-drug to provide the desired anti-cancer effects.[000104] In some embodiments, the pro-drug is administered parenterally, e.g., intravenously, intraperitonially, intratumorally, or subcutaneously.[000105] The dosage amounts of the pro-drug may range from about 1 to about 200 mg of patient body weight. In some embodiments, the dosage (effective amount) of the pro-drug is about 1 mg / kg, 2 mg / kg, about 3 mg / kg, 4 mg / kg, 5 mg / kg, about 7.5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 50 mg / kg, about 75 mg / kg, about 100 mg / kg, about 125 mg / kg, about 150 mg / kg, about 175 mg / kg, or about 200 mg / kg. In some embodiments, the dosage amount of the pro-drug may range from about 10 to about 75 mg / kg. In some embodiments, the dosage amount of the pro-drug may range from about 10 to about 40 mg / kg. In some embodiments, the dosage amount of the pro-drug is about 10 mg / kg, infused over the course of 1 hour every three weeks.[000106] In some embodiments, the dosage amount of the pro-drug is chosen to achieve a concentration of about 0.1 to about 1 μM in the blood.[000107] The pro-drug is administered the subject after any unbound antibody-enzyme conjugate is cleared from the subject’s circulatory system, at least to an acceptable level. The body clears biological actives such as antibody-enzyme conjugates in specific and / or dose-dependent ways. Specific clearance may rely on target mediated drug disposition in which the interaction between the antibody of the antibody-enzyme conjugate and the antibody’s target contributes significantly to the kinetics of antibody-enzyme conjugate distribution and clearance. Specific clearance may rely on a non-saturable dose. Nonspecific clearance refers to target independent, nonspecific cellular uptake of the antibody-enzyme conjugate by pinocytosis / proteolysis and / or proteolytic degradation and subsequent removal from circulation. See, Malik and Ghatol, “Understanding How Monoclonal Antibodies Work” in StatPearls, StatPearls Publishing, Treasure Island, Florida21AFSDOCS:304317774.1VIA EFS Attorney Docket No. 044797.00005 Date of Deposit: December 22, 2025(2023); Wang etal., Clin. Pharmacol. Ther. 84(5):548-58 (2008); and Ovacik and Lin, Clin. Transl. Sci. 11(6):540-552 (2018).[000108] In some embodiments, the in vivo level of the antibody-enzyme conjugate is measured before the administration of the pro-drug is commenced. Methods of measuring the in vivo levels of the antibody-enzyme conjugate are known in the art and include antibody detection, radiolabeling, and enzymatic activity. In some embodiments, the level of the antibody-enzyme conjugate is determined by toxicity studies of the activated drug (i.e., cleaved pro-drug). In some embodiments, the level of antibody-enzyme conjugate is measured in a peripheral blood sample. In some embodiments, the administration of the pro-drug is commenced once the level of the antibody-enzyme conjugate falls below a pre-determined value. In some embodiments, the level of the antibody-enzyme conjugate is between about 25 and about 50 percent of the original antibodyenzyme conjugate level.[000109] In some embodiments, the administration of the pro-drug is commenced from about 24 to about 96 hours after administration of the antibody-enzyme conjugate. In some embodiments, the time between antibody-enzyme conjugate administration and pro-drug administration is about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 52 hours, about 60 hours, about 72 hours, about 84 hours, about 96 hours, about 108 hours, or about 120 hours. In some embodiments, the intravenous administration of the pro-drug is commenced about 24 to about 120 hours after intravenous administration of the antibody-enzyme conjugate. In some embodiments, the administration (intravenously or intratumorally) of the pro-drug is commenced about 1 to about 24 hours after intratumoral administration of the antibody-enzyme conjugate.[000110] The time taken for the concentration of an antibody in the blood to decrease to half its original concentration (i.e., by clearance) is referred to as an antibody’s half-life. In some embodiments, pro-drug administration is commenced after about 0.5 half-life of the antibodyenzyme conjugate, about 1 half-life of the antibody-enzyme conjugate, about 1.5 half-lives of the antibody-enzyme conjugate, or about 2 half-lives of the antibody-enzyme conjugate. Methods of determining half-lives of antibody conjugates are known in the art.[000111] In some embodiments, the antibody-enzyme conjugate therapy entails administering a total of about 1 to about 6 doses (e.g., 2 doses, 3 doses, 4 doses, 5 doses, or 6 doses) of the antibodyenzyme conjugate. In some embodiments, the antibody-enzyme conjugate therapy entails 22AFSDOCS:304317774.1VIA EFS Attorney Docket No. 044797.00005 Date of Deposit: December 22, 2025administration of about 1 dose per day, about 2 doses per day, about 3 doses per day, or about 4 doses per day.[000112] In some embodiments, the pro-drug therapy entails administering a total of about 1 to about 6 doses (e.g., 2 doses, 3 doses, 4 doses, 5 doses, or 6 doses) of the pro-drug. In some embodiments, the pro-drug therapy entails administration of about 1 dose per day, about 2 doses per day, about 3 doses per day, or about 4 doses per day. In some embodiments, the pro-drug is administered in a single dose.[000113] The treatment may involve one or more cycles of administration of ADEPT agents. The term “cycle” when used herein to refer to a treatment regimen, refers to the administration of the antibody-enzyme conjugate followed by the administration of the pro-drug. In some embodiments, the treatment cycle includes administering the antibody-enzyme conjugate therapy in an infusion of antibody-enzyme conjugate over 120 minutes, measuring the level of antibody-enzyme conjugate in peripheral blood samples, and administering the pro-drug in a course of three bolus injections, each injection one hour apart, once the level of antibody-enzyme conjugate in the peripheral blood samples falls below a predetermined level. In some embodiments, the cycle may be repeated once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every eight weeks, or once every ten weeks.[000114] The cycles may be repeated over a period of time. In some embodiments, the cycles are repeated for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 2 years, or about 3 years.[000115] In some embodiments, the cycle is repeated every week, every two weeks or every three weeks for a treatment period of about one month to about three months.Kits[000116] Pharmaceutical compositions may be assembled into a kit or pharmaceutical system for use in treating cancer. The kits or pharmaceutical systems may include one or more dosage formulations containing an antibody-enzyme conjugate and a pharmaceutically acceptable carrier disposed in a suitable container, e.g., tube, vial, ampoule, bottle, syringe, or bag. In some embodiments, the kit or pharmaceutical system may also include one or more dosage formulations of the pro-drug. The pro-drug is formulated separately and is disposed in a separate container from 23AFSDOCS:304317774.1VIA EFS Attorney Docket No. 044797.00005 Date of Deposit: December 22, 2025the antibody-enzyme conjugate. The kits or pharmaceutical systems of the disclosure may also comprise printed instructions for using the active(s) contained therein.Combination Therapy[000117] In some embodiments, the present methods may include co-administration of another anti-cancer therapy. The term “co-administered” includes substantially contemporaneous administration, by the same or separate dosage forms, or sequentially, e.g., as part of the same treatment regimen or by way of successive treatment regimens. Thus, if given sequentially, at the onset of administration of the second therapy, the first of the two therapies may still be detectable at effective concentrations at the site of treatment. The sequence and time interval may be determined such that they can act together (e.g., synergistically to provide an increased benefit than if they were administered otherwise). For example, the therapeutics may be administered at the same time or sequentially in any order at different points in time; however, if not administered at the same time, they may be administered sufficiently close in time so as to provide the desired therapeutic effect, which may be in a synergistic fashion. Thus, the terms are not limited to the administration of the active agents exactly at the same time.[000118] In some embodiments, the additional anti-cancer therapy is chemotherapy, radiotherapy (e.g., with temozolomide (TMZ) (Temodar®)), immunotherapy, targeted therapy, pro-apoptotic therapy, or cell cycle regulation therapy, therapy with thalidomide, lenalidomide, bortezomib, and / or melphalan.[000119] In some embodiments, the additional anti-cancer therapy is surgical resection. Surgical resection may include a surgical imaging technique (e.g., intraoperative magnetic resonance imaging (MRI), diffusion tensor imaging, awake craniotomy, cortical mapping, stereotactic guidance, and fluorescent-guided resection), which facilitate the delineation of tumor borders and optimizes maximal safe surgical resection (Wilson et al., Surg. Neurol. Int. 5:64 (2014)). Surgical resection may be performed with a surgical microscope or exoscope, preferably equipped with filters to image the surgical imaging technique. In some embodiments, the surgical resection is subtotal resection, near-total resection, gross-total resection, maximal safe resection, or supratotal resection (Gerritsen et al., Neurooncol. Pract. 9(5):364-379 (2022)).[000120] In some embodiments, surgical resection is performed before any other anti-cancer therapy (e.g., ADEPT treatment, radiotherapy, and / or chemotherapy). In some embodiments,24AFSDOCS:304317774.1VIA EFS Attorney Docket No. 044797.00005 Date of Deposit: December 22, 2025surgical resection is performed after ADEPT therapy but before any additional anti -cancer therapy (e.g., radiotherapy and / or chemotherapy). In some embodiments, surgical resection is performed after all other anti-cancer therapy.Synthesis of the Prodrug 3-cyclopentyl-a-phenylalanine-methotrexate[000121] A further aspect of the present invention is directed to a method of synthesizing 3- cyclopentyl-a-phenylalanine-methotrexate (IUPAC: N-(4-(((2,4-Diamino-6- pteridinyl)methyl)methylamino)benzoyl)-L-glutam-l-yl-3-cyclopentyl-L-phenylalanine). As illustrated in Fig. 13, the method may be conducted in as few as 7 steps. Unless indicated to the contrary, reactants (including the first reactant), solvents and catalysts are commercially available. For example, in addition to solvents and catalysts, reactants such as Boc-3-bromo-D-phenylalanine, cyclopentyl-boronic acid, (S)-2-(((benzyloxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanoic acid (Z-Glu(OtBu)-OH), DAMP A,[000122] A first step entails treating (reacting) Boc-3-bromo-D-phenylalanine with a solution of tert-butyl, 2,2,2-trichloroacetimidate (TBTA) which yields tc / 7-butyl 3-(3-bromophenyl)-2-(( / c / 7- butoxycarbonyl)amino)propanoate. In some embodiments, the first step may entail treating (reacting) Boc-3-bromo-D-phenylalanine with a solution of tert-butyl, 2,2,2-trichloroacetimidate (TBTA) in a mixture of tetrahydrofuran (THF) and dichloromethane (DCM), followed by extraction in an organic solvent, and washing, which yields tert-butyl 3-(3-bromophenyl)-2-((tert- butoxycarbonyl)amino)propanoate (shown as compound 1 in Fig. 13).[000123] A second step entails mixing a solution of the product of step 1 (i.e., compound 1) with cyclopentyl-boronic acid, palladium (II) acetate, and triphenyl phosphine in a solvent mixture, which yields tert-butyl 2-((tert-butoxycarbonyl)amino)-3-(3-(cyclopent-l-en-l- yl)phenyl)propanoate (shown as compound 2 in Fig. 13). In some embodiments, the mixing may be conducted in an organic solvent mixture.[000124] A third step entails removing the Boc protecting group from compound 2, which yields the free amine (shown as compound 3 in Fig. 13).[000125] A fourth step entails reacting the free amine produced in step 3 (i.e., compound 3) with (S)-2-(((benzyloxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanoic acid (Z-Glu(OtBu)-OH) using a peptide coupling agent, in the presence of 1 -hydroxybenzotriazole (HOBt) and N, N- Diisopropylethylamine (DIEA), which yields tert-Butyl N-((benzyloxy)carbonyl)-5-O-tert-butyl- 25AFSDOCS:304317774.1VIA EFS Attorney Docket No. 044797.00005 Date of Deposit: December 22, 2025L-glutam-l-yl-3-cyclopentyl-L-phenylalaninate (shown as compound 4 in Fig. 13). In some embodiments, the reacting may be conducted in an organic solvent or mixture thereof.[0001261 A fifth step entails reduction of the double bond on the cyclopentene ring and deprotection of the benzyloxycarbonyl (Cbz) group on the reaction product of step 4 (i.e., compound 4), which yields tert-Butyl 5-O-tert-butyl-L-glutam-l-yl-3-cyclopentyl-L-phenylalaninate (shown as compound 5 in Fig. 13).[000127] A sixth step entails reacting the product of step 5 (the free amine which is compound 5) with deoxyaminopteroic acid (DAMP A) which yields tert-Butyl 5-O-tert-butyl-N-(4-(((2,4- diamino-6-pteridinyl)methyl)methylamino )benzoyl)-L-glutam-l-yl-3-cyclopentyl-L- phenylalaninate (shown as compound 6 in Fig. 13).[000128] A seventh step entails treating the peptide produced in step 6 (i.e., compound 6) to HC1 gas, followed by neutralization to pH 7, to yield N-(4-(((2,4-Diamino-6- pteridinyl)methyl)methylamino)benzoyl)-L-glutam-l-yl-3-cyclopentyl-L-phenylalanine (shown as compound 7 in Fig. 13).[000129] Modifications with respect to choices of solvents, etc., and selection of suitable amounts and volumes of reactants and solvents, and determination of suitable reaction parameters (e.g., time, temperature and pressure) are well within the level of skill in the art. Illustrative and nonlimiting modifications and selections are described in the following paragraphs below, as well as in Example 4, in connection with each step.[000130] In some embodiments, the treating in the first step may be conducted for about 3 days at room temperature, the extraction may be conducted with ethyl acetate, the washing may be conducted with a solution of potassium carbonate, and / or the product of step 1 may be purified using standard techniques such as automatic column chromatography (Hex / EtOAc). Separately or in addition, the first step may entail treating Boc-3-bromo-D-phenylalanine in a suitable amount, e.g., about 4 g (0.019 mol) with a solution of tert-butyl, 2,2,2-trichloroacetimidate (TBTA) at a suitable volume, e.g., about 1.04 mb, in a mixture of tetrahydrofuran (THF) at a suitable volume, e.g., about 2 mL.[000131] In some embodiments, the solvent mixture used in the second step includes toluene and ethanol, e.g., at a 9:1 ratio. The mixture of the reactants and the solvents may be stirred under conditions of suitable time, temperature and pressure, e.g., about 120° C for about 18 hours under inert atmosphere. The product of step 2 may be purified using standard techniques such as 26AFSDOCS:304317774.1VIA EFS Attorney Docket No. 044797.00005 Date of Deposit: December 22, 2025automatic column chromatography (Hex / EtOAc). Separately or in addition, the second step may entail mixing the solution of the product of step 1 in a suitable amount, e.g., about 0.767 g (0.00192 mol) with cyclopentyl-boronic acid in a suitable amount, e.g., about 0.343 g (0.00307 mol), palladium (II) acetate in a suitable amount, e.g., about 0.043 g (0.000192 mol), K2CO3 in a suitable amount, e.g., about 1.38 g (0.00651 mol) and triphenyl phosphine in a suitable amount, e.g., about 0.05 g (0.000192 mol), in the solvent mixture.[000132] In some embodiments, the third step of removing the Boc protecting group is conducted using 2,2,2-Trifluoroacetic acid (TFA) / DCM (e.g., in a suitable ratio, e.g., about 1:4), as is known in the art. The product of step 3 may be purified using standard techniques.[000133] In some embodiments, the peptide coupling agent used in the fourth step is Hexafluorophosphate Benzotriazole Tetramethyl Uronium (HBTU) and the organic solvent is DMF. The reaction mixture may be stirred under conditions of suitable time and temperature, e.g., overnight at about room temperature. The product of step 4 may be purified by known techniques such as automatic column chromatography (DCM / MeOH). Separately or in addition, the fourth step may entail reacting the free amine produced in step 3 in a suitable amount, e.g., about 0.250 g (0.000870 mol) with (S)-2-(((benzyloxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanoic acid (Z- Glu(OtBu)-OH) in a suitable amount, e.g., about 0.196 g (0.000580 mol), in the organic solvent at a suitable volume, e.g., about 30 mb, in the presence of HBTU as the peptide coupling agent in a suitable amount, e.g., about 0.330 g (0.000870 mol), in the presence of 1 -hydroxybenzotriazole (HOBt) in a suitable amount, e.g., about 0.118 g (0.000870 mol), and N, N-Diisopropylethylamine (DIEA) at a suitable volume, e.g., about 0.253 mL.[000134] In some embodiments, the reduction of the double bond on the cyclopentene ring and the deprotection in the fifth step entail pressurizing a mixture of the product of step 4 (i.e., compound 4) in a suitable amount, e.g., about 0.356g (0.00597 mol) and methanol at a suitable volume, e.g., about 28 ml, in the presence of 10% palladium on carbon (Pd / C) in a suitable amount, e.g., about 0.069 g, with hydrogen at a suitable pressure, e.g., about 1 bar, for a suitable time period, e.g., about 4 hours, at about room temperature. The product of step 5 may be purified by known techniques such as filtration over a pad of celite.[000135] In some embodiments, the sixth step entails reacting the product of step 5 (the free amine, shown as compound 5 in Fig. 13) with deoxyaminopteroic acid (DAMP A) using HBTU, HOBt, and DIEA in DMF, under conditions of suitable temperature, e.g., at about room temperature and 27AFSDOCS:304317774.1VIA EFS Attorney Docket No. 044797.00005 Date of Deposit: December 22, 2025suitable time, e.g., about 18 hours. The reacting may be followed by extraction, e.g., with ethyl acetate (EtOAc), and washing, e.g., with sodium hydroxide (NaOH) and brine. The product of step 6 (i.e., compound 6 shown in Fig. 13) may be purified using standard techniques such as automatic column chromatography (DCM / MeOH). Separately or in addition, the sixth step may entail reacting the product of step 5 (the free amine) in a suitable amount, e.g., about 0.0174 g (0.00366 mol) with deoxyaminopteroic acid (DAMP A) in a suitable amount, e.g., about 0.079 g (0.000244 mol) using HBTU in a suitable amount, e.g., about 0.139 g (0.000366 mol), HOBT in a suitable amount, e.g., about 0.049 g (0.000366 mol), and DIEA at a suitable volume, e.g., about 0.206 mL, in DMF at a suitable volume, e.g., about 9 mL.[000136] In some embodiments, the seventh step entails dissolving the peptide produced in step 6 (compound 6 as shown in Fig. 13) in a nitromethane solution and wherein the treating with HC1 gas is conducted at about room temperature for about one hour. Upon neutralization to pH 7, the precipitate may be filtered and washed, allowing the recovery of pure N-(4-(((2,4-Diamino-6- pteridinyl)methyl)methylamino)benzoyl)-L-glutam-l-yl-3-cyclopentyl-L-phenylalanine, i.e., 3- cyclopentyl-a-phenylalanine-methotrexate. Separately or in addition, the seventh step may entail dissolving a suitable amount, e.g., about 0.075g (0.0960 mmol) of compound 6 (the peptide produced in step 6) in a suitable volume, e.g., about 7 ml of the nitromethane solution.[000137] These and other aspects of the present application will be further appreciated upon consideration of the following Examples, which are intended to illustrate certain embodiments of the application but are not intended to limit its scope, as defined by the claims.EXAMPLESExample 1: Making and purifying hCPA T268G.[000138] The active site of hCPA may be mutated to enable its enzymatic cleavage of the pro-drug (3-cyclopentyl-alpha-phenylalanine-methotrexate) to the cytotoxic drug methotrexate. To produce the mutant hCPA, the appropriate point mutation of T268G of CPA was created using site-directed mutagenesis of the pCMV3 plasmid, commercially available from Sino Biological (Cat # HG10504-CH) and altering the appropriate nucleic acids to convert threonine (T) at the 28AFSDOCS:304317774.1VIA EFS Attorney Docket No. 044797.00005 Date of Deposit: December 22, 2025corresponding amino acid position 268 to a glycine (G) by changing the codon from ACC to GGC. This was performed in a vector containing human CPA 1 with a histidine tag at the C-terminus, The histidine was placed at the C-terminus as the N-terminus is cleaved from the protein during cell processing when the ER signal sequence is removed after hCPA is shuttled through the secretory pathway.[000139] HEK cells were transfected with either WT hCPA (NCBI Ref. No. NM 001868.2) or mutant hCPA plasmids and for the over-production of the respective protein. It was confirmed by western blot that wild-type and mutant hCPA is indeed secreted from these cells. The majority of the hCPA was found in the media and very little wild-type or mutant hCPA was found in the cellular lysate (FIG. 2). Using hygromycin resistance encoded in the hCPA plasmid, stable lines expressing and secreting hCPA were created and cryogenically stored.[000140] Both WT and mutant CPA were isolated using immobilized metal affinity chromatography (IMAC) Nickel columns (HisPur Ni-NTA spin columns; Thermofisher), according to normal purification protocols (FIG. 3). Detection by western blot of the eluted column demonstrates that hCPA was isolated in the first three elutions off the nickel column. The isolated protein is substantially pure as demonstrated by the Coomassie staining (FIG. 3, left) where only the brightest bands correspond to hCPA with some minor bands above and below that may represent activated (lower) or aggregate hCPA. Again, there is no substantial hCPA in the cell lysates, even after attempted purification.[000141] Because hCPA is initially expressed as a pro-enzyme that does not actively degrade unfolded proteins within the ER, golgi, or cell secretory system, the hCPA must be activated by removal of the pro-domain; a process that is similar to in vivo cleavage in the digestive system. 'Trypsin cleaves the pro-domain in vivo, so the conditions for activating hCPA using trypsin in vitro were optimized. A trypsin dose of 5 pg / mL was deemed optimal for activating CPA, as observed by the emergence of the smaller, active hCPA bands lacking the pro-domain (FIG. 4). Trypsin concentrations above 5 pg / mL degraded CPA as a whole, likely by non-specific cleavage throughout the protein, as evidenced by a lack of bands on the western blot (FIG 4). The optimal duration of activation was also tested and was determined to be about 5 hours (FIG. 5).[000142] Finally, another activation test was performed to determine if 15 pg / mE of trypsin could activate hCPA within an hour. After the one-hour incubation, trypsin was inactivated by the immediate addition of the activated hCPA solution to the cells in media containing fetal bovine 29AFSDOCS:304317774.1VIA EFS Attorney Docket No. 044797.00005 Date of Deposit: December 22, 2025serum (FBS), which inactivates trypsin (FIG. 6). If the activated hCPA was not used immediately, it was either frozen and stored until thawing immediately prior to use in cells with media with FBS, or in a small amount of media with FBS to quench the trypsin activity.Example 2: Identification and selection of CD206 (MMR) as a target for ADEPT.[000143] Many different markers of M2 / TAMs in rat brain tumors were investigated. The goal was to find markers that were specifically expressed on macrophages within the tumor microenvironment (z.e., TAMs) but not on microglia, the normal resident brain macrophages. After investigating over a dozen key markers of M2 macrophages, the immunohistochemical data demonstrated that the mannose macrophage receptor (MMR, also known as CD206) was specifically expressed by macrophages within the brain tumors. MMR is a marker produced at consistently high levels by TAMs. It is associated with M2 macrophages as well but is currently the main marker used to identify TAMs in various aggressive tumors (see Table 1 for a partial list of cancers with known MMR+macrophages). In glioblastomas, resident macrophages are activated to express MMR, and MMR-expressing macrophages were only found within the tumor microenvironment (FIGs. 7A - 7B). Thus, MMR was identified as a viable tumor-specific marker for ADEPT targeting.[000144] Next, it was determined if MMR was a specific and viable target in human glioblastoma tissue. Post-mortem malignant glioblastoma tissues were obtained from three patients and normal brain tissue from two of the same patients as normal brain controls. Unfortunately, because of the difficulty in antigen presentation from these samples, reliable staining from only one tissue and resulting control was obtained. However, it was clear that MMR staining was observed in the tissue specimen from the malignant brain tumor and no MMR was observed in the normal brain despite the expected presence of macrophages / microglia. Thus, MMR appears to be upregulated in TAMs associated with human tumors and it is a viable, specific target for TAMs in human glioblastoma (FIGs. 8 - 9). Because the TAMs are non-cancerous, targeting MMR on normal, non-cancerous TAMs allows for targeting many different cancers that have infiltrating TAMs (and therefore MMR+cells).Example 3: Identifying an enzyme and pro-drug combination[000145] For ADEPT to be effective, the pro-drug must be selectively activated by the enzyme linked to the tumor-targeted antibody (i.e., the antibody of the antibody-enzyme conjugate). The 30AFSDOCS:304317774.1VIA EFS Attorney Docket No. 044797.00005 Date of Deposit: December 22, 2025pro-drug should be stable, inactive, and not cleaved (i.e., activated) by endogenous enzymes. This creates a problem in that when a naturally occurring human enzyme is used, the pro-drug will be activated by any tissue where the human enzyme is normally expressed. If a foreign enzyme is used as a strategy to prevent activation by endogenous enzymes, then rejection by the immune system becomes a major issue, as occurred in the early clinical trials using CPG2 (Bagshawe and Sharma, Transplant. Proc. 28(6)3156-3158 (1996)). To be effective, the enzyme must remain stably bound to the antibody long enough for the antibody to bind and target the tumor, for unbound antibodies to be cleared by the body, and for the pro-drug to subsequently be administered and encounter the enzyme at the tumor site.[000146] To circumvent this issue, point mutations in the human carboxypeptidase A (hCPA) enzyme were identified that could be coupled with various pro-drug forms of methotrexate (Smith et al., J. Biol. Chem. 272(25): 15804-15816 (1997)). Methotrexate is an inhibitor of the folate pathway that ultimately results in prevention of nucleotide synthesis, thus preventing cells from dividing and ultimately causing cell death. It is currently approved for breast cancer, epidermoid cancers of the head and neck, advanced mycosis fungoides (cutaneous T cell lymphoma), nonHodgkin’s lymphomas, and lung cancer, particularly squamous cell and small cell types. However, it is also associated with bone marrow suppression, hepatic or renal dysfunction, gastrointestinal distress, mucocutaneous damage, and neurotoxicity. These side effects can be life threatening (Pannu, Curr. Drug Metab. 20(9):714-719 (2019)). Thus, any strategy that utilizes the beneficial aspects of methotrexate, while limiting its toxicity, would be beneficial to a large number of cancer indications.[000147] hCPA has an active site that fits amino acids with large side chains, for example, phenylalanine. 3-cyclopentyl-alpha-phenylalanine-methotrexate has a pro-drug moiety, 3- cyclopentyl-alpha-phenylalanine, that is too large to fit in the active site of wild type-hCPA (FIG.10). Thus, the pro-drug is unable to be activated by the body’s endogenously expressed hCPA. However, a single point mutation in the gene for carboxypeptidase A increases the size of the active site, allowing the phenylalanine-containing pro-drug to fit only in the mutated hCPAl (mCPAl), while the single point mutation should not lead to rejection by the immune system (FIG. 11). Example 4: Synthesis of 3-cyclopentyl-a-phenylalanine-methotrexate31AFSDOCS:304317774.1VIA EFS Attorney Docket No. 044797.00005 Date of Deposit: December 22, 2025[000148] As shown in Fig. 13, in a first step, commercially available Boc-3-bromo-D- phenylalanine was treated with a solution of tris((1-benzyl-4-triazolyl)methyl)amine (TBTA) in a mixture of tetrahydrofuran (THF) and dichloromethane (DCM) for 3 days at room temperature. The crude material was extracted with ethyl acetate and washed with a solution of potassium carbonate before being purified using automatic column chromatography (Hex / EtOAc) to yield the tert-butyl 3-(3-bromophenyl)-2-((tert-butoxycarbonyl)amino)propanoate in 55% yield.[000149] For the second step, commercially available cyclopentyl-boronic acid, palladium (II) acetate, and triphenyl phosphine were added to a solution of the pure compound from step 1 in toluene and ethanol at a 9:1 ratio. The mixture was stirred at 120° C for 18 hours under inert atmosphere. Upon completion of the reaction, the crude material was purified by automatic column chromatography (Hex / EtOAc) to yield tert-butyl 2-((tert-butoxycarbonyl)amino)-3-(3-(cyclopent- l-en-l-yl)phenyl)propanoate in 77% yield.[000150] The Boc protecting group was removed using the standard 2,2,2-Trifluoroacetic acid (TFA) / DCM (at a 1:4 ratio) method. Upon completion of the reaction and subsequent workup of the crude material, the pure tert-butyl 2-amino-3-(3-(cyclopent-l-en-l-yl)phenyl)propanoate was recovered in 74% yield.[000151] The free amine was then reacted with commercially available (S)-2- (((benzyloxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanoic acid (Z-Glu(OtBu)-OH) in DMF using O-(benzotriazol-l-yl)-N, N, N', N'-tetramethyluronium hexafluorophosphate (HBTU) as a peptide coupling agent in the presence of 1 -hydroxybenzotriazole (HOBt) and N, N- Diisopropylethylamine (DIEA). After stirring the mixture overnight at room temperature, the crude was purified by automatic column chromatography (DCM / MeOH) to yield tert-Butyl N- ((benzyloxy)carbonyl)-5-O-tert-butyl-L-glutam-l-yl-3-cyclopentyl-L-phenylalaninate in 77% yield.[000152] The next step used hydrogen on carbon to reduce the double bond on the cyclopentene ring and deprotect the benzyloxy carbonyl (Cbz) group. A methanol mixture containing the product obtained from the previous step and 10% palladium on carbon (Pd / C) was pressurized with hydrogen at 1 bar for 4 hours at room temperature. Upon filtration over a pad of celite, the pure tert-Butyl 5-O-tert-butyl-L-glutam-l-yl-3-cyclopentyl-L-phenylalaninate was obtained in 58% yield.32AFSDOCS:304317774.1VIA EFS Attorney Docket No. 044797.00005 Date of Deposit: December 22, 2025[000153] The free amine was reacted with the commercially available deoxyaminopteroic acid (DAMPA) using HBTU, HOBt, DIEA in DMF at room temperature for 18 hours. The crude material was then extracted with ethyl acetate (EtOAc) and washed with sodium hydroxide (NaOH) and brine before being purified by automatic column chromatography (DCM / MeOH) to yield the tert-Butyl 5-O-tert-butyl-N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino )benzoyl)-L- glutam-l-yl-3-cyclopentyl-L-phenylalaninate in 42% yield.[000154] The final compound was obtained by treating the peptide dissolved in a nitromethane solution to HC1 gas at room temperature for one hour. Upon neutralization to pH 7, the precipitate was filtered and washed, allowing the recovery of pure N-(4-(((2,4-Diamino-6- pteridinyl)methyl)methylamino)benzoyl)-L-glutam-l-yl-3-cyclopentyl-L-phenylalanine in 56% yield.[000155] Overall, the synthesis of the pure N-(4-(((2,4-Diamino-6- pteridinyl)methyl)methylamino)benzoyl)-L-glutam-l-yl-3-cyclopentyl-L-phenylalanine took 7 steps from commercially available starting material.Example 5: Activity of 3-cyclopentyl-alpha-phenylalanine-methotrexate.[000156] A cell viability assay using 3-[4,5,dimethylthiazol-2-yl]-5-[3-carboxymethoxy-phenyl]- 2-[4-sulfophenyl]-2H -tetrazolium (MTS) was performed using a standard curve of known amounts of live HT1080 human tumor cells, with increasing numbers of live tumor cells incubated with increasing doses of methotrexate (MTX) or the pro-drug 3-cyclopentyl-alpha-phenylalanine- methotrexate (labeled as MTX / Phe in FIG. 12). As can be observed in FIG. 12, the pro-drug is inactive at doses beyond 20-fold higher than those at which active methotrexate has maximum activity. The IC50of methotrexate was about 0.02 pg / mL, while the pro-drug remained non-toxic at all doses tested (up to 1 pg / mL). This assay was completed 3 more times with very similar outcomes.[000157] All patent publications and non-patent publications are indicative of the level of skill of those skilled in the art to which this disclosure pertains. All these publications are herein incorporated by reference to the same extent as if each individual publication were specifically and individually indicated as being incorporated by reference.[000158] Although the disclosure herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles 33AFSDOCS:304317774.1VIA EFS Attorney Docket No. 044797.00005 Date of Deposit: December 22, 2025and applications of the present disclosure. It is therefore to be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present disclosure as defined by the appended claims.34AFSDOCS:304317774.1

Claims

VIA EFS Attorney Docket No. 044797.00005 Date of Deposit: December 22, 2025What is claimed is:

1. A method of treating a cancer characterized by the presence of a solid tumor, comprising:administering to a human patient in need thereof a first agent comprising an antibody or fragment thereof that binds a CD206 receptor (mannose macrophage receptor; MMR) present on tumor associated macrophages (TAMs), conjugated to a mutant of human carboxypeptidase A (hCPA) wherein the mutant contains a glycine residue at position 268 in place of threonine (hCPA T268G); followed byadministering to the patient a second agent comprising 3-cyclopentyl-alpha-phenylalanine-methotrexate.

2. The method of claim 1, wherein the cancer is glioblastoma.

3. The method of claim 1, wherein the cancer is colorectal cancer.

4. The method of claim 1, wherein the cancer is gastric cancer.

5. The method of claim 1, wherein the cancer is head and neck cancer.

6. The method of claim 1, wherein the cancer is hepatocellular cancer.

7. The method of claim 1, wherein the cancer is prostate cancer.

8. The method of any one of claims 1-7, wherein the first agent is administered intravenously, intraperitonially, intratumorally, or subcutaneously.

9. The method of any one of claims 1-8, wherein the dosage amount of the first agent is administered as an intravenous infusion over a period of time comprising about 15 to about 120 minutes.35AFSDOCS:304317774.1VIA EFS Attorney Docket No. 044797.00005 Date of Deposit: December 22, 202510. The method of any one of claims 1-9, wherein the dosage amount of the first agent achieves a concentration of about 0.05 to about 1 pM at a tumor site in the patient.

11. The method of any one of claims 1-10, wherein the second agent is administered intravenously, intraperitonially, intratumorally, or subcutaneously.

12. The method of any one of claims 1-11, wherein the dosage amount of the second agent is about 10 mg / kg, infused over the course of 1 hour every three weeks.

13. The method of any one of claims 1-11, wherein the dosage amount of the second agent achieves a concentration of about 0.1 to about 1 pM in the blood of the patient.

14. The method of any one of claims 1-13, wherein the administration of the first agent and the administration of the second agent is repeated every week, every two weeks, or every three weeks.

15. The method of claim 14, wherein the administration of the first agent and the administration of the second agent is conducted over a period of about 1 month to about three months.

16. The method of claim 2, wherein the first and second agents are administered to the human patient following surgical resection.

17. A system for treating a cancer characterized by the presence of a solid tumor, comprising: a first agent comprising an antibody or fragment thereof that binds a CD206 receptor (mannose macrophage receptor; MMR) present on tumor associated macrophages (TAMs), conjugated to a mutant of human carboxypeptidase A (hCPA) wherein the mutant contains a glycine residue at position 268 in place of threonine (hCPA T268G); anda second agent comprising 3-cyclopentyl-alpha-phenylalanine-methotrexate.

18. The system of claim 17, wherein the hCPA T268G has the amino acid sequence of SEQ ID NO: 37.36AFSDOCS:304317774.1VIA EFS Attorney Docket No. 044797.00005 Date of Deposit: December 22, 202519. The system of claim 17 or 18, wherein the first agent and the second agent are disposed in separate containers.

20. The system of claim 19, wherein the separate containers are disposed in a kit.

21. A method of synthesizing 3-cyclopentyl-a-phenylalanine-methotrexate, comprising:(i) treating Boc-3-bromo-D-phenylalanine with a solution of tris((1-benzyl-4-triazolyl)methyl)amine (TBTA) in a mixture of tetrahydrofuran (THF) and dichloromethane (DCM), followed by extraction in an organic solvent, and washing, which yields tert-butyl 3- (3-bromophenyl)-2-((tert-butoxycarbonyl)amino)propanoate;(ii) mixing a solution of the product of (i) with cyclopentyl-boronic acid, palladium (II) acetate, and triphenyl phosphine, in an organic solvent or mixture thereof, which yields tert- butyl 2-((tert-butoxycarbonyl)amino)-3-(3-(cyclopent-l-en-l-yl)phenyl)propanoate;(iii) removing the Boc protecting group which yields a free amine;(iv) reacting the free amine produced in (iii) with (S)-2-(((benzyloxy)carbonyl)amino)-5- (tert-butoxy)-5-oxopentanoic acid (Z-Glu(OtBu)-OH) in the presence of a peptide coupling agent, 1 -hydroxybenzotriazole (HOBt) and N, N-Diisopropylethylamine (DIEA), which yields tert-Butyl N-((benzyloxy)carbonyl)-5-O-tert-butyl-L-glutam-l-yl-3-cyclopentyl-L- phenylalaninate;(v) reducing the double bond on the cyclopentene ring and deprotecting the benzyloxycarbonyl (Cbz) group on the compound produced in (iv), which yields tert-Butyl 5- O-tert-butyl-L-glutam-l-yl-3-cyclopentyl-L-phenylalaninate;(vi) reacting the compound produced in (v) with deoxyaminopteroic acid (DAMP A), which yields the peptide tert-Butyl 5-O-tert-butyl-N-(4-(((2,4-diamino-6- pteridinyl)methyl)methylamino)benzoyl)-L-glutam-l-yl-3-cyclopentyl-L-phenylalaninate; and (vii) treating the peptide produced in (vi) to HC1 gas, followed by neutralization to pH 7, which yields N-(4-(((2,4-Diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-l-yl-3- cyclopentyl-L-phenylalanine (3-cyclopentyl-a-phenylalanine-methotrexate).37AFSDOCS:304317774.1

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