Redirecting cytotoxic T cells with chemically programmed antibodies

Redirecting cytotoxic T cells with chemically programmed antibodies

Bioorg. Med. Chem. 28 (2020) 115834 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry journal homepage:

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Bioorg. Med. Chem. 28 (2020) 115834

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry journal homepage:

Redirecting cytotoxic T cells with chemically programmed antibodies Junpeng Qi *, Christoph Rader * Department of Immunology and Microbiology, The Scripps Research Institute, Jupiter, FL 33458, USA



Keywords: Chemical programming Bispecific antibodies Chimeric antigen receptors T cells Cancer cell surfaceome

T-cell engaging bispecific antibodies (T-biAbs) mediate potent and selective cytotoxicity by combining speci­ ficities for target and effector cells in one molecule. Chemically programmed T-biAbs (cp-T-biAbs) are precisely assembled compositions of (i) small molecules that govern cancer cell surface targeting with high affinity and specificity and (ii) antibodies that recruit and activate T cells and equip the small molecule with confined bio­ distribution and longer circulatory half-life. Conceptually similar to cp-T-biAbs, switchable chimeric antigen receptor T cells (sCAR-Ts) can also be put under the control of small molecules by using a chemically pro­ grammed antibody as a bispecific adaptor molecule. As such, cp-T-biAbs and cp-sCAR-Ts can endow small molecules with the power of cancer immunotherapy. We here review the concept of chemically programmed antibodies for recruiting and activating T cells as a promising strategy for broadening the utility of small mol­ ecules in cancer therapy.

1. Chemically programmed antibodies A conventional discrimination of therapeutic small and large mole­ cules in cancer is that the former predominantly serve for targeting intracellular and the latter for targeting extracellular proteins. None­ theless, an increasing number of small molecules that bind to cancer cell surface receptors have been identified. In fact, it has become clear that many extracellular proteins harbor thermodynamically and kinetically favored pockets for binding small molecules with high affinity and specificity. These pockets are often too small to reach or to distinguish by monoclonal antibodies (mAbs). Examples include members of the G protein-coupled receptor (GPCR)1, solute carrier (SLC)2, and ion chan­ nel superfamilies of transmembrane proteins to which generating large molecules, in particular mAbs, has been difficult3. Despite various campaigns to generate small molecules to every protein4,5, along with the availability of increasingly complex chemical libraries, such as DNAencoded libraries (DELs) that can be selected on intact cells6, the pocketome fraction of the cancer cell surfaceome has remained vastly underexplored and underutilized. This is in part due to the pharmaco­ kinetic and pharmacodynamic shortcomings of small molecules compared to mAbs, specifically their unobstructed biodistribution, short circulatory half-life, and inability to recruit effector proteins and cells of the immune system. Based on these considerations, the concept of chemically programmed antibodies was borne7. Chemically pro­ grammed antibodies are hybrid molecules designed to merge the

pharmacological advantages of small molecules and antibodies and assembled with stoichiometric precision8. By molecularly defined co­ valent conjugation to an antibody molecule, the biodistribution of a cancer cell surface targeting small molecule is confined to the extra­ cellular (intravascular and extravascular) fluid, its circulatory half-life increases several orders of magnitude, and it is endowed with the abil­ ity to recruit effector proteins and cells of the innate immune system to mediate complement-dependent cytotoxicity (CDC), antibodydependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP). Typically, chemically programmed anti­ bodies consist of a variable small molecule component and an invariable antibody component. Chemical programming of the expensive invari­ able antibody component with a virtually unlimited variety of inex­ pensive small molecule components makes this modular platform highly versatile and economically attractive compared to conventional mAbs8. Here we review the application of the concept of chemically pro­ grammed antibodies to adaptor systems that are able to recruit effector cells of the adaptive immune system. This includes T-cell engaging bispecific antibodies (T-biAbs) and switchable chimeric antigen receptor T cells (sCAR-Ts), both of which can be put under the control of a var­ iable small molecule component. Collectively, chemically programmed T-biAbs (cp-T-biAbs) and chemically programmed sCAR-Ts (cp-sCARTs) provide small molecules that selectively target the cancer cell sur­ face access to the power of cancer immunotherapy.

* Corresponding author. E-mail addresses: [email protected] (J. Qi), [email protected] (C. Rader). Received 11 September 2020; Received in revised form 20 October 2020; Accepted 24 October 2020 Available online 2 November 2020 0968-0896/© 2020 Elsevier Ltd. All rights reserved.

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Bioorganic & Medicinal Chemistry 28 (2020) 115834

2. First-generation cp-T-biAbs

Thus, T-biAbs expand the scope of T cell-mediated killing from MHCpresented peptides (T cell antigens) to native cell surface receptors (B cell antigens). The first and currently only T-biAb approved by the FDA (2014) and EMA (2015) is blinatumomab (Blincyto®), a CD19 × CD3 tandem single-chain variable fragment (scFv) for the treatment of re­ fractory or relapsed (r/r) pre-B acute lymphoblastic leukemia (pre-BALL)10. As reviewed elsewhere11,12, numerous conventional T-biAbs are currently in preclinical and clinical investigations for the therapy of hematologic and solid malignancies. This assortment includes both TbiAbs that are, like blinatumomab, based on antibody fragments with short circulatory half-life and T-biAbs that resemble the ~ 150-kDa immunoglobulin G (IgG) format of natural antibodies and most

Conventional T-biAbs consist of two antibody arms of which one binds to a T cell and the other to a cancer cell surface receptor. The T cell surface receptor is commonly CD3, specifically its CD3ε chain, a signal transducing component of the T cell receptor (TCR) complex (Fig. 1). Similar to the natural interaction of the TCR with a major histocom­ patibility complex (MHC)-presented peptide, T-biAbs crosslink effector and target cell and promote the formation of an immunological synapse. The immunological synapse is also known as cytolytic synapse as concomitant and coordinated release of cytotoxic granules containing perforin and granzymes by the effector cell results in target cell lysis9.

Fig. 1. Concept and architecture of cp-T-biAbs and cp-sCAR-Ts. Antibody heavy and light chain domains are shown in gray and white, respectively. The three complementarity-determining regions of the variable domains are depicted as small ovals. (A–F) Different formats of cp-T-biAbs. The CD3-binding site of cp-T-biAbs is displayed by the variable heavy (dark blue) and light (light blue) chain domain. This T cell engaging antibody component is site-specifically conjugated to a small molecule component (orange star) that binds a cancer cell surface receptor (orange). As such, cp-T-biAbs crosslink T cell and cancer cell and promote the formation of cytolytic synapses. (G, H) Different formats of bispecific adaptor molecules of cp-sCAR-Ts. A universal CAR (blue) on a CAR-T cell is bound by a recombinantly fused (G) or chemically conjugated (H) peptide (blue triangle) that is covalently connected to a cancer cell surface receptor-binding small molecule (orange star). Catalytic antibody h38C2 serves as carrier of the small molecule in D-F and as carrier of the small molecule and peptide in G and H. 2

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Bioorganic & Medicinal Chemistry 28 (2020) 115834

therapeutic mAbs13,14. Unlike conventional T-biAbs, cp-T-biAbs bind to a cancer cell surface receptor via a small molecule that is covalently conjugated to a T-cell engaging antibody. The concept of attaching a cancer cell-binding ligand to a T-cell engaging antibody was first published by Liu et al.15 based on random chemical conjugation of a 13-amino acid peptide to a mouse anti-human CD3 mAb. The peptide was an analog of α-melano­ cyte stimulating hormone (MSH) and as such enabled the redirection of cytotoxic T cells to the MSH receptor on melanoma cells. A similar approach was pursued by Zhou et al.16 for an antagonistic synthetic peptide targeting gastrin-releasing peptide receptor (GRPR), a GPCR expressed on small cell lung cancer (SCLC) cells. A GRPR × CD3 antibody-peptide conjugate was assembled by random chemical conju­ gation and shown to mediate T-cell dependent killing of SCLC cell lines in in vitro and in vivo models. Applying this concept to a small molecule rather than a peptide, Kranz et al.17 used folate as the cancer cell-binding ligand. Random chemical conjugation of folate to various TCR/CD3-targeting mAbs yielded first-generation cp-T-biAbs that mediated the killing of folate receptor 1 (FOLR1)-expressing cancer cells in the presence of T cells at picomolar concentrations in vitro. In subsequent studies, the same group generated FOLR1 × TCR cp-T-biAbs in scFv18 and antigen-binding fragment (Fab) format19 which reduce the molecular weight from ~ 150 kDa to ~ 25 kDa and ~ 50 kDa, respectively, and as such may have pharmacological advantages when treating solid malignancies. Both scFv and Fab-based FOLR1 × TCR cp-T-biAbs revealed potent in vivo activity against FOLR1-expressing tumors grown in mice with endoge­ nous T cells19,20. As natural small molecule, folate, also known as vitamin B9, has been a preferred chemical component for establishing proof-of-concept of first- and second-generation cp-T-biAbs. FOLR1 is a glycosylphosphatidylinositol (GPI)-anchored glycoprotein that binds folate with nanomolar affinity and mediates its transmembrane trans­ port via receptor-mediated endocytosis. Its overexpression in various solid malignancies including ovarian and lung cancer has made FOLR1 an attractive target for both small molecule- and antibody-based diag­ nostic and therapeutic reagents21,22.

anti-human CD3 Fab with an engineered unnatural amino acid residue, para-acetyl-phenylalanine (pAcPhe), encoded by the stop codon UAG in the constant domain of the heavy chain fragment (Fig. 1B). The trans­ lational incorporation of pAcPhe requires a pair of ectopically expressed nonhost tRNA and tRNA-aminoacyl-tRNA synthetase (e.g., an engi­ neered Methanocaldococcus jannaschii tRNATyr/tyrosyl-tRNA synthetase pair) that is acting orthogonally to the translational machinery of the host (e.g., Escherichia coli)27. The unique ketone group of E. coli-pro­ duced Fab-pAcPhe was conjugated to an aminooxy derivative of folate to afford a stable oxime linkage. The resulting FOLR1 × CD3 cp-T-biAbs revealed potent and selective in vitro and in vivo activity28. Using the same antibody component and conjugation chemistry, Kim et al29 used 2-[3-(1,3-dicarboxypropyl)ureidopentanedioic acid (DUPA) as small molecule component for assembling cp-T-biAbs that target prostate cancer cells. DUPA binds to prostate-specific membrane antigen (PSMA) with nanomolar affinity30. Interestingly, the location of the conjugation site did not affect PSMA+ prostate cancer cell binding but was critical for T-cell dependent potent and selective in vitro cytotoxicity. Thus, a key feature of the Fab-pAcPhe antibody component is the ability to incorporate the unnatural amino acid residue at various positions, affording the testing of multiple bispecific compositions for optimal orientation and spacing which is critical for the efficient formation and duration of the cytolytic synapse. This is an advantage over conventional PSMA × CD3 T-biAbs which may be limited by a more rigid architecture. In the same study, a bivalent version was also investigated by intro­ ducing pAcPhe at two different positions in the constant domains of heavy chain fragment and light chain29. The (PSMA)2 × CD3 cp-T-biAb was superior to the corresponding PSMA × CD3 cp-T-biAbs in terms of in vitro cytotoxicity, mediated T-cell dependent potent and selective killing of xenografted PSMA+ prostate cancer cell lines in immunodeficient mice, and revealed a circulatory half-life of ~ 6 h in normal rats. Notably, an improved version based on these cp-T-biAbs, CCW702, was recently translated to a phase I clinical trial in metastatic castration resistant prostate cancer ( Identifier: NCT04077021). Both Fab-Sec and Fab-pAcPhe are antibody components with an engineered noncanonical amino acid that displays unique chemical reactivity, i.e. selenol and ketone functionality, respectively, not found among the 20 canonical amino acids. Site-specific assemblies of anti­ body and small molecule components can also be built with canonical amino acids that display unique chemical reactivity. For example, se­ lective reduction of the Fab’s interchain disulfide bridge connecting heavy chain fragment and light chain, affords two thiol functionalities that can be utilized for site-specific conjugation. Patterson et al.31 generated a PSMA × CD3 cp-T-biAb by conjugating a dibromomalei­ mide derivative of DUPA to an enzymatically generated anti-human CD3 Fab with reduced interchain disulfide bridge following treatment with dithiothreitol. Notably, the formation of a dithiomaleimide bridge restored the covalent linkage of heavy chain fragment and light chain (Fig. 1C). In the presence of human peripheral blood mononuclear cells (PBMC), this PSMA × CD3 cp-T-biAb mediated subnanomolar cytotox­ icity against PSMA+ but not PSMA- prostate cancer cell lines. Canonical amino acids of antibodies can also have unique chemical reactivity due to their location. This is the case for the reactive Lys residue of catalytic antibody 38C2 and its humanized version h38C2 which is located at the bottom of an 11-Å deep hydrophobic haptenbinding site32,33. The ε-amino group of the reactive Lys residue has a highly perturbed pKa of 6.0 that renders it unprotonated at physiolog­ ical pH and highly nucleophilic. As such, it can be utilized for chemical programming with small molecules derivatized with 1,3-diketone or β-lactam haptens7,34. Various h38C2-peptide conjugates assembled by chemical programming were clinically translated8. More recently, chemical programming of catalytic antibody h38C2 has also been applied to the generation of cp-T-biAbs. Walseng et al.35 combined the variable domains of h38C2 and the variable domains of a humanized anti-human CD3 mAb in a ~ 55-kDa disulfide-bridged diabody format (Fig. 1D) that is known as DART36. By chemical programming with a

3. Second-generation cp-T-biAbs Second-generation cp-T-biAbs assemble antibody and small mole­ cule component site-specifically rather than randomly, yielding stoi­ chiometrically defined hybrid molecules. As such, second-generation cpT-biAbs manifest the idea of chemically programming an invariable antibody molecule at a uniquely reactive conjugation site with a vari­ able small molecule. Examples of second-generation cp-T-biAbs are shown in Fig. 1A-F. Cui et al.23 used a humanized anti-human CD3 Fab with an engi­ neered selenocysteine (Sec) residue at the C-terminus of the heavy chain fragment as antibody component (Fab-Sec) (Fig. 1A). As the 21st natural amino acid24, Sec is encoded by the stop codon UGA and in mammalian cells translationally incorporated by a Sec incorporation sequence (SECIS) in the 3′ UTR of the mRNA. Taking advantage of the different pKa value of the selenol group (pKa 5.2) compared to the thiol group of Cys (pKa 8.3), the Fab-Sec was site-specifically conjugated with maleimide-derivatized small molecules including folate and LLP2A. LLP2A is a peptidomimetic derived from a one-bead-one-compound chemical library for binding to activated human integrin α4β1 with high affinity and specificity25. Integrins constitute another popular proof-of-concept cancer cell surface receptor for cp-T-biAbs due to their ability to bind tripeptide motifs such as Arg-Gly-Asp (integrin αvβ3 and others) and Leu-Asp-Val (integrin α4β1) which have been mimicked by numerous small molecules26. The anti-CD3 Fab-Sec chemically pro­ grammed with folate and LLP2A, respectively, revealed potent cyto­ toxicity against FOLR1+ and integrin α4β1+ cancer cell lines and primary cancer cells in the presence of primary T cells23. A structurally similar second-generation cp-T-biAb used a mouse 3

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β-lactam hapten derivative of folate, the DART was converted to a FOLR1 × CD3 cp-T-biAb and mediated T-cell dependent potent and selective killing of FOLR1+ ovarian cancer cell lines in vitro and in vivo. Expanding this assembly strategy to Fc-containing cp-T-biAbs, Qi et al.37 engineered two asymmetric antibody components consisting of a hu­ manized anti-human CD3 scFv arm and a single (Fig. 1E) or double h38C2 Fab arm (Fig. 1F). Following chemical programming with a β-lactam hapten derivative of folate, FOLR1 × CD3 and (FOLR1)2 × CD3 cp-T-biAb acquired binding to FOLR1+ ovarian cancer cell lines, enabled crosslinking of T cells, and revealed potent and selective activity in vitro and in vivo. Notably, the bivalent version consistently out­ performed the monovalent version with respect to all three criteria. To avoid systemic T-cell activation through Fcγ receptor (FcγR)-mediated display of the humanized anti-human CD3 scFv arm, an aglycosylated Fc domain was used that does not impact neonatal Fc receptor (FcRn)mediated recycling. Overall, irrespective of these various molecular assemblies, the invariable antibody component of cp-T-biAbs serves as both carrying and targeting moiety and the variable small molecule component serves as the other targeting moiety. As such, cp-T-biAbs are highly modular; the same protein can be chemically programmed with a virtually un­ limited diversity of specificities. By contrast, conventional T-biAbs require a new protein for every new specificity. Furthermore, spacing the two specificities, which is important for cytolytic synapse formation, is more restricted in conventional T-biAbs compared to cp-T-biAbs.

that binds with picomolar affinity to a 14-amino acid peptide derived from yeast transcription factor GCN450. In the absence of the Fabpeptide switch, the anti-GCN4 peptide CAR-T stays inactive. In the presence of the Fab-peptide switch and cancer cells expressing the Fab’s antigen, such as CD19, CD20, or HER2, the anti-GCN4 peptide CAR-T gets activated. Alternatively, the bispecific adapter molecule may combine a variable biological component and an invariable chemical component, for example a Fab-small molecule conjugate with an engi­ neered unnatural amino acid residue interface, such as para-azidophe­ nylalanine (pAzPhe). Site-specific conjugation of the pAzPhe residue to fluorescein isothiocyanate (FITC) derivatized with a cyclooctyne func­ tionality affords a Fab-FITC switch that binds to a human anti-FITC scFvbased universal CAR49,51. Using a Fab-peptide or Fab-small molecule switch has the advantage that the site of recombinant or chemical conjugation of peptide or small molecule, respectively, can be varied for optimal cytolytic synapse formation. In addition, incorporation of two peptides or small molecules enables bivalent engagement of the uni­ versal CAR-T. Collectively, side-by-side comparisons of optimized sCARTs with conventional CAR-Ts revealed similar potency and selectivity in vitro and in vivo48,49,51. In addition to these two examples, several more sCAR-T platforms have been developed and translated to clinical trials46. By splitting targeting and signaling modules, sCAR-Ts not only afford an additional layer of control over activity and toxicity but also the ability to simul­ taneously or sequentially target different cancer cell surface receptors. This can be achieved by combining two or more bispecific adaptor molecules of different specificities. Furthermore, the resting period be­ tween switch dosing promotes memory, expansion, and persistence of sCAR-Ts52. The concept of a bispecific adapter molecule has allowed to apply chemical programming to CAR-Ts by combining an invariable antibody component to a variable small molecule component. Qi et al. recently described the first cp-sCAR-T platform53. This system uses the universal CAR that binds to the GCN4 peptide. Catalytic antibody h38C2 was recombinantly equipped with the GCN4 peptide at the N- or C-terminus of heavy chain fragment or light chain and chemically programmed with a β-lactam hapten derivative of folate. As such, it served as bispecific adaptor molecule bridging FOLR1 on ovarian cancer cell lines to the anti-GCN4 peptide CAR-Ts (Fig. 1G). The same study showed that a set of trifunctional compounds consisting of GCN4 peptide, folate, and β-lactam hapten groups spaced by polyethylene glycol (PEG) linkers of variable lengths allows to chemically program both cancer cell and universal CAR-T engagement (Fig. 1H). Both cp-sCAR-Ts revealed potent and selective in vitro and in vivo activity against FOLR1+ ovarian cancer cell lines which was strictly dependent on chemically program­ ming the switch. Notably, the length of the PEG linker impacted CAR-T activity, suggesting that the spacing of CAR-T and cancer-cell engaging groups requires optimization. It is conceivable that the additional flex­ ibility afforded by introducing the GCN4 peptide by chemical conjuga­ tion (Fig. 1H) compared to recombinant fusion (Fig. 1G) is advantageous for tailoring and fine tuning optimal cytolytic synapse formation. A bifunctional compound consisting of GCN4 peptide and folate groups alone was shown to also serve as bispecific adaptor molecule but its much shorter circulatory half-life rendered it inferior to the chemically programmed Fab in a mouse model of ovarian cancer. Taken together, the sCAR-T platform that uses a Fab-peptide switch48 was expanded from a conventional Fab to a chemically programmed Fab in order to broaden its utility to cancer cell surface receptors that can be targeted by small molecules53. As discussed for cp-T-biAbs and in contrast to conventional bispecific adapter molecules, chemically programmed bispecific adaptor mole­ cules have the advantage of (i) requiring only one protein for essentially any specificity and (ii) affording an unrestricted spacing of cancer cell receptor-targeting and universal CAR-T engaging modules. The ability to simultaneously or sequentially target different cancer cell surface receptors by combining or alternating different chemically programmed

4. cp-sCAR-Ts Conceptually similar to T-biAbs, CAR-T cells constitute another promising class of cancer immunotherapeutics38,39. Three CD19targeting CAR-Ts have received FDA approval thus far; (i) tisagenle­ cleucel (Kymriah®; Novartis) for the treatment of patients up to 25 years old with r/r pre-B-ALL in 2017 and adult patients with r/r diffuse large B-cell lymphoma (DLBCL) in 201840,41; (ii) axicabtagene ciloleucel (Yescarta®; Gilead) for the treatment of adult patients with r/r DLBCL in 201741,42; and (iii), in 2020, brexucabtagene autoleucel (Tecartus®; Gilead) for the treatment of adult patients with r/r mantle cell lym­ phoma43. CAR-Ts are built by transducing autologous T cells from cancer patients with CARs that fuse an extracellular antibody fragment, typically scFv, to a transmembrane segment followed by the cytoplasmic signaling domain of a T cell costimulatory receptor (typically CD28 or 41BB) and the cytoplasmic signaling domain of CD3ζ of the TCR complex. As such, a CAR links antibody-mediated binding to T-cell activation44. Compared to T-biAbs, CAR-Ts face two key challenges. First, as patienttailored therapy, the production and administration of CAR-Ts is logis­ tically challenging. It involves the collection, activation, transduction, expansion, cryopreservation, and infusion of autologous T cells45. Sec­ ond, as living drugs, CAR-Ts can persist forever in the cancer patient. The FDA-approved CAR-Ts do not have on/off switches that permit control over their on-target-on-tissue, on-target-off-tissue, and offtarget-off-tissue activities and carry the risk of cytokine release syn­ drome (CRS) and immune effector cell-associated neurotoxicity syn­ drome (ICANS). Gaining control over these adverse events by advanced CAR-T engineering and CAR-T target discovery has become a major effort in this field. One approach is based on sCAR-Ts, which are recruited and activated by a bispecific adapter molecule that serves as on/off switch46,47. This system is based on a universal CAR that does not bind cancer cell surface receptors on its own but only through the bis­ pecific adaptor molecule (Fig. 1). The latter engages CAR and cancer cell surface receptor with an invariable and a variable, respectively, chem­ ical or biological component. An example of a bispecific adapter molecule consisting of two bio­ logical components is a Fab (variable biological component) that binds to a cancer cell surface receptor and displays a recombinantly fused peptide (invariable biological component) that binds to a universal CAR48,49. Specifically, Rodgers et al.48 based the universal CAR on a scFv 4

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bispecific adaptor molecules makes the cp-sCAR-T platform particularly attractive for the treatment of heterogeneous solid malignancies which are notorious for refraction and relapse.

2 Wang WW, Gallo L, Jadhav A, Hawkins R, Parker CG. The druggability of solute carriers. J Med Chem. 2020;63:3834–3867. 3 Oprea TI, Bologa CG, Brunak S, et al. Unexplored therapeutic opportunities in the human genome. Nat Rev Drug Discov. 2018;17:317–332. 4 Mullard A. A probe for every protein. Nat Rev Drug Discov. 2019;18:733–736. 5 Carter AJ, Kraemer O, Zwick M, Mueller-Fahrnow A, Arrowsmith CH, Edwards AM. Target 2035: probing the human proteome. Drug Discov Today. 2019;24:2111–2115. 6 Cai B, Kim D, Akhand S, et al. Selection of DNA-encoded libraries to protein targets within and on living cells. J Am Chem Soc. 2019;141:17057–17061. 7 Rader C, Sinha SC, Popkov M, Lerner RA, Barbas 3rd CF. Chemically programmed monoclonal antibodies for cancer therapy: adaptor immunotherapy based on a covalent antibody catalyst. Proc Natl Acad Sci USA. 2003;100:5396–5400. 8 Rader C. Chemically programmed antibodies. Trends Biotechnol. 2014;32:186–197. 9 de la Roche M, Asano Y, Griffiths GM. Origins of the cytolytic synapse. Nat Rev Immunol. 2016;16:421–432. 10 Kantarjian H, Stein A, Gokbuget N, et al. Blinatumomab versus chemotherapy for advanced acute lymphoblastic leukemia. N Engl J Med. 2017;376:836–847. 11 Labrijn AF, Janmaat ML, Reichert JM, Parren P. Bispecific antibodies: a mechanistic review of the pipeline. Nat Rev Drug Discov. 2019;18:585–608. 12 Rader C. Bispecific antibodies in cancer immunotherapy. Curr Opin Biotechnol. 2020; 65:9–16. 13 Rader C. “One, if by land, and two, if by sea”: bispecific antibodies join the revolution. Methods. 2019;154:1–2. 14 Wu X, Demarest SJ. Building blocks for bispecific and trispecific antibodies. Methods. 2019;154:3–9. 15 Liu MA, Nussbaum SR, Eisen HN. Hormone conjugated with antibody to CD3 mediates cytotoxic T cell lysis of human melanoma cells. Science. 1988;239:395–398. 16 Zhou J, Chen J, Zhong R, Mokotoff M, Shultz LD, Ball ED. Targeting gastrin-releasing peptide receptors on small cell lung cancer cells with a bispecific molecule that activates polyclonal T lymphocytes. Clin Cancer Res. 2006;12:2224–2231. 17 Kranz DM, Patrick TA, Brigle KE, Spinella MJ, Roy EJ. Conjugates of folate and antiT-cell-receptor antibodies specifically target folate-receptor-positive tumor cells for lysis. Proc Natl Acad Sci U S A. 1995;92:9057–9061. 18 Cho BK, Roy EJ, Patrick TA, Kranz DM. Single-chain Fv/folate conjugates mediate efficient lysis of folate-receptor-positive tumor cells. Bioconjug Chem. 1997;8: 338–346. 19 Rund LA, Cho BK, Manning TC, Holler PD, Roy EJ, Kranz DM. Bispecific agents target endogenous murine T cells against human tumor xenografts. Int J Cancer. 1999;83: 141–149. 20 Roy EJ, Cho BK, Rund LA, Patrick TA, Kranz DM. Targeting T cells against brain tumors with a bispecific ligand-antibody conjugate. Int J Cancer. 1998;76:761–766. 21 Xia W, Low PS. Folate-targeted therapies for cancer. J Med Chem. 2010;53: 6811–6824. 22 Fernandez M, Javaid F, Chudasama V. Advances in targeting the folate receptor in the treatment/imaging of cancers. Chem Sci. 2018;9:790–810. 23 Cui H, Thomas JD, Burke Jr TR, Rader C. Chemically programmed bispecific antibodies that recruit and activate T cells. J Biol Chem. 2012;287:28206–28214. 24 Hatfield DL, Gladyshev VN. How selenium has altered our understanding of the genetic code. Mol Cell Biol. 2002;22:3565–3576. 25 Peng L, Liu R, Marik J, Wang X, Takada Y, Lam KS. Combinatorial chemistry identifies high-affinity peptidomimetics against alpha4beta1 integrin for in vivo tumor imaging. Nat Chem Biol. 2006;2:381–389. 26 Zhao J, Santino F, Giacomini D, Gentilucci L. Integrin-targeting peptides for the design of functional cell-responsive biomaterials. Biomedicines. 2020;8. 27 Young TS, Schultz PG. Beyond the canonical 20 amino acids: expanding the genetic lexicon. J Biol Chem. 2010;285:11039–11044. 28 Kularatne SA, Deshmukh V, Gymnopoulos M, et al. Recruiting cytotoxic T cells to folate-receptor-positive cancer cells. Angew Chem Int Ed Engl. 2013;52:12101–12104. 29 Kim CH, Axup JY, Lawson BR, et al. Bispecific small molecule-antibody conjugate targeting prostate cancer. Proc Natl Acad Sci U S A. 2013;110:17796–17801. 30 Kularatne SA, Venkatesh C, Santhapuram HK, et al. Synthesis and biological analysis of prostate-specific membrane antigen-targeted anticancer prodrugs. J Med Chem. 2010;53:7767–7777. 31 Patterson JT, Isaacson J, Kerwin L, et al. PSMA-targeted bispecific Fab conjugates that engage T cells. Bioorg Med Chem Lett. 2017;27:5490–5495. 32 Barbas 3rd CF, Heine A, Zhong G, et al. Immune versus natural selection: antibody aldolases with enzymic rates but broader scope. Science. 1997;278:2085–2092. 33 Rader C, Turner JM, Heine A, et al. A humanized aldolase antibody for selective chemotherapy and adaptor immunotherapy. J Mol Biol. 2003;332:889–899. 34 Gavrilyuk JI, Wuellner U, Barbas 3rd CF. Beta-lactam-based approach for the chemical programming of aldolase antibody 38C2. Bioorg Med Chem Lett. 2009;19: 1421–1424. 35 Walseng E, Nelson CG, Qi J, et al. Chemically programmed bispecific antibodies in diabody format. J Biol Chem. 2016;291:19661–19673. 36 Johnson S, Burke S, Huang L, et al. Effector cell recruitment with novel Fv-based dual-affinity re-targeting protein leads to potent tumor cytolysis and in vivo B-cell depletion. J Mol Biol. 2010;399:436–449. 37 Qi J, Hymel D, Nelson CG, Burke Jr TR, Rader C. Conventional and chemically programmed asymmetric bispecific antibodies targeting folate receptor 1. Front Immunol. 2019;10:1994. 38 June CH, O’Connor RS, Kawalekar OU, Ghassemi S, Milone MC. CAR T cell immunotherapy for human cancer. Science. 2018;359:1361–1365. 39 Sadelain M, Riviere I, Riddell S. Therapeutic T cell engineering. Nature. 2017;545: 423–431.

5. Outlook The discussed examples depicted in Fig. 1 reveal a new class of therapeutics at the interface of chemistry and immunology. The chem­ ical programming of T-biAbs and sCAR-Ts with small molecules unlocks a vast targetable and druggable space on the cancer cell surfaceome to the power of cancer immunotherapy. This largely uncharted territory is currently still dominated by small molecule binding sites on cancer cell surface receptors that can also be targeted by conventional T-biAbs and CAR-Ts, including integrin α4β1, FOLR1, and PSMA in the discussed examples. Receptors that have proven elusive for conventional antibody-based cancer therapy, such as GPCRs, SLCs, and ion channels constitute unexplored therapeutic opportunities3 that could be partic­ ularly attractive for small molecules equipped with the pharmacokinetic and pharmacodynamic properties of antibodies and antibody fragments. What makes small molecules suitable for chemical programming is different from traditional small molecule drug discovery. First, their conjugation to an antibody or antibody fragment fundamentally changes their pharmacological properties in terms of absorption, distribution, metabolism, and excretion (ADME). There is also no need for oral bioavailability and cell membrane permeability. Second, chemical pro­ gramming of the antibody or antibody fragment requires a functionality that affords derivatization with a linker without affecting affinity and specificity of the pharmacophore. It also requires aqueous solubility and stability of the small molecule. Third, the binding properties of the small molecule should resemble the high affinity (0.1–100 nM) and specificity of antibodies. Both should be validated after chemical programming using biochemical and immunological assays that are well established for antibodies. Fourth, its endowment with T-cell engagement renders the small molecule independent of any antagonistic or agonistic activity. However, as the pocketome of the cancer cell surfaceome includes functional small molecule binding sites for antagonizing enzymes and receptor-ligand interactions54–56, additive or synergistic mechanisms of action mediated by small molecule and antibody component are also conceivable. Based on an ever increasing database of high-resolution structural information of cell surface receptors driven by advances in X-ray crystallography and cryogenic electron microscopy, computa­ tional tools for the identification of small molecule binding sites have been developed54,57,58. Using molecular docking, virtual chemical li­ braries are screened for small molecules that fit these pockets59,60. In addition to these in silico efforts, the exploration of the pocketome by high throughput screening of increasingly complex real chemical li­ braries is well underway61–63. With cp-T-biAb and cp-sCAR-T platforms at hand, virtual and real hits can now be probed for their ability to re­ cruit and activate T cells for potent and selective eradication of cancer cells, vastly expanding the cancer targetome. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this review. Acknowledgments This review was supported by National Institutes of Health (NIH) grants R01 CA174844, R01 CA181258, and R01 CA204484. References 1 Hutchings CJ. A review of antibody-based therapeutics targeting G protein-coupled receptors: an update. Expert Opin Biol Ther. 2020;20:925–935.


J. Qi and C. Rader

Bioorganic & Medicinal Chemistry 28 (2020) 115834 52 Viaud S, Ma JSY, Hardy IR, et al. Switchable control over in vivo CAR T expansion, B cell depletion, and induction of memory. Proc Natl Acad Sci USA. 2018;115: E10898–E10906. 53 Qi J, Tsuji K, Hymel D, et al. Chemically programmable and switchable CAR-T therapy. Angew Chem Int Ed Engl. 2020;59:12178–12185. 54 Bhagavat R, Sankar S, Srinivasan N, Chandra N. An augmented pocketome: detection and analysis of small-molecule binding pockets in proteins of known 3D structure. Structure. 2018;26:499–512 e492. 55 Kufareva I, Ilatovskiy AV, Abagyan R. Pocketome: an encyclopedia of small-molecule binding sites in 4D. Nucleic Acids Res. 2012;40:D535–D540. 56 Xu D, Jalal SI, Sledge GW, Meroueh SO. Small-molecule binding sites to explore protein-protein interactions in the cancer proteome. Mol Biosyst. 2016;12: 3067–3087. 57 Volkamer A, Griewel A, Grombacher T, Rarey M. Analyzing the topology of active sites: on the prediction of pockets and subpockets. J Chem Inf Model. 2010;50: 2041–2052. 58 Ghersi D, Sanchez R. Beyond structural genomics: computational approaches for the identification of ligand binding sites in protein structures. J Struct Funct Genomics. 2011;12:109–117. 59 Kitchen DB, Decornez H, Furr JR, Bajorath J. Docking and scoring in virtual screening for drug discovery: methods and applications. Nat Rev Drug Discov. 2004;3: 935–949. 60 Abel R, Mondal S, Masse C, et al. Accelerating drug discovery through tight integration of expert molecular design and predictive scoring. Curr Opin Struct Biol. 2017;43:38–44. 61 Gerry CJ, Schreiber SL. Chemical probes and drug leads from advances in synthetic planning and methodology. Nat Rev Drug Discov. 2018;17:333–352. 62 Goodnow Jr RA, Dumelin CE, Keefe AD. DNA-encoded chemistry: enabling the deeper sampling of chemical space. Nat Rev Drug Discov. 2017;16:131–147. 63 Neri D, Lerner RA. DNA-encoded chemical libraries: a selection system based on endowing organic compounds with amplifiable information. Annu Rev Biochem. 2018;87:479–502.

40 O’Leary MC, Lu X, Huang Y, et al. FDA approval summary: tisagenlecleucel for treatment of patients with relapsed or refractory B-cell precursor acute lymphoblastic leukemia. Clin Cancer Res. 2019;25:1142–1146. 41 Chow VA, Shadman M, Gopal AK. Translating anti-CD19 CAR T-cell therapy into clinical practice for relapsed/refractory diffuse large B-cell lymphoma. Blood. 2018; 132:777–781. 42 Bouchkouj N, Kasamon YL, de Claro RA, et al. FDA approval summary: axicabtagene ciloleucel for relapsed or refractory large B-cell lymphoma. Clin Cancer Res. 2019;25: 1702–1708. 43 Wang M, Munoz J, Goy A, et al. KTE-X19 CAR T-cell therapy in relapsed or refractory mantle-cell lymphoma. N Engl J Med. 2020;382:1331–1342. 44 Gross G, Waks T, Eshhar Z. Expression of immunoglobulin-T-cell receptor chimeric molecules as functional receptors with antibody-type specificity. Proc Natl Acad Sci USA. 1989;86:10024–10028. 45 Kohl U, Arsenieva S, Holzinger A, Abken H. CAR T cells in trials: recent achievements and challenges that remain in the production of modified T cells for clinical applications. Hum Gene Ther. 2018;29:559–568. 46 Minutolo NG, Hollander EE, Powell Jr DJ. The emergence of universal immune receptor T cell therapy for cancer. Front Oncol. 2019;9:176. 47 Feldmann A, Arndt C, Koristka S, Berndt N, Bergmann R, Bachmann MP. Conventional CARs versus modular CARs. Cancer Immunol Immunother. 2019;68: 1713–1719. 48 Rodgers DT, Mazagova M, Hampton EN, et al. Switch-mediated activation and retargeting of CAR-T cells for B-cell malignancies. Proc Natl Acad Sci U S A. 2016; 113:E459–E468. 49 Cao Y, Rodgers DT, Du J, et al. Design of switchable chimeric antigen receptor T cells targeting breast cancer. Angew Chem Int Ed Engl. 2016;55:7520–7524. 50 Zahnd C, Spinelli S, Luginbuhl B, Amstutz P, Cambillau C, Plückthun A. Directed in vitro evolution and crystallographic analysis of a peptide-binding single chain antibody fragment (scFv) with low picomolar affinity. J Biol Chem. 2004;279: 18870–18877. 51 Ma JS, Kim JY, Kazane SA, et al. Versatile strategy for controlling the specificity and activity of engineered T cells. Proc Natl Acad Sci USA. 2016;113:E450–E458.