1 (1975); Hongo et al., Hybridoma, 14 (3): 253-260 (1995), Harlow et al., Antibodies: A laboratory Manual, (Cold Spring Harbor Laboratory Press, 2^nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas5b2-bZ] (Elsevier, N.Y., 1981)), recombinant DNA methods (see, e.g. U.S. Pat. No. 4,816,567), phage-display technologies (see, e.g., Clatkson et al., Nature, 352: 624-628 (1991); Marks et al., J. Mol. BiolTll-. 581-597 (1992); Sidhu et al., 7. Mo! Bio! W>(2}-. 299-310 (2004); Lee et al., J. Mol. Biol.3W{5}. 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. «£4101(34): 12467-12472 (2004); and Lee et al., /. Immunol. Methods 284(1 -2): 119-132 (2004), and technologies for producing human or human-like antibodies in animals that have parts or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., WO 1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741; Jakobovits et al., Proc. Natl. Acad. Sci. (ISAM: 2551 (1993); Jakobovits et a\., Nature 362: 255-258 (1993); Bruggemann et al., Year in ImmunolJM (1993); U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and U.S. Pat. No. 5,661,016; Marks et al., Bio/TechnologyW. 779-783 (1992); Lonberg et al., Nature363: 856-859 (1994); Morrison, Nature^-. 812-813 (1994); Fishwild et al., Nature Biotechnoi U: 845-851 (1996); Neuberger, Nature BiotechnoL 14: 826 (1996); and Lonberg and Huszar, intern. Rev. Immunol. 13: 65-93 (1995).

The terms “full-length antibody”, “intact antibody”, or “whole antibody” are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antibody fragment. Specifically, full-length 4-chain antibodies include those with heavy and light chains including an Fc region. Full-length heavy-chain only antibodies include the heavy chain (such as VHH) and an Fc region. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. In some cases, the intact antibody may have one or more effector functions.

An “antibody fragment” comprises a portion of an intact antibody, preferably the antigen binding and/or the variable region of the intact antibody. Examples of antibody fragments include, but are not limited to Fab, Fab', F(ab')₂ and Fv fragments,- diabodies,- linear antibodies (see U.S. Pat. No.5,641,870, Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062) [1995]; singlechain antibody molecules; single-domain antibodies [such as VHH), and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produced two identical antigen-binding fragments, called “Fab” fragments, and a residual “Fc” fragment, a designation reflecting the ability to crystallize readily. The Fab fragment consists of an entire L chain along with the variable region domain of the H chain (V_H), and the first constant domain of one heavy chain (C_H1 ). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an antibody yields a single large F(ab')₂ fragment which roughly corresponds to two disulfide linked Fab fragments having different antigen-binding activity and is still capable of cross-linking antigen. Fab' fragments differ from Fab fragments by having a few additional residues at the carboxy-terminus of the C_H1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab’ in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab '^antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

The Fc fragment comprises the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of antibodies are determined by sequences in the Fc region, the region which is also recognized by Fc receptors (FcR) found on certain types of cells.

The term “constant domain” refers to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable domain, which contains the antigen-binding site. The constant domain contains the C_H 1 , C_H2 and C_H3 domains (collectively, CH) of the heavy chain and the CHL (or CL) domain of the light chain. The “light chains” of antibodies (immunoglobulins) from any mammalian species can be assigned to one of two clearly distinct types, called kappa (“x”) and lambda (“k”), based on the amino acid sequences of their constant domains.

“Fv” is the minimum antibody fragment which contains a complete antigen-recognition and -binding site. This fragment consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (3 loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

“Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibody fragments that comprise the V_H and V_L antibody domains connected into a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the V_H and V_L domains which enables the sFv to form the desired structure for antigen binding. For a review of the sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp.269-315 (1994).

“Functional fragments” of the antibodies described herein comprise a portion of an intact antibody, generally including the antigen binding or variable region of the intact antibody or the Fc region of an antibody which retains or has modified FcR binding capability. Examples of antibody fragments include linear antibody, single-chain antibody molecules and multispecific antibodies formed from antibody fragments.

The term “diabodies” refers to small antibody fragments prepared by constructing sFv fragments (see preceding paragraph) with short linkers (about 5-10 residues) between the V_H and V_L domains such that inter-chain but not intra-chain pairing of the V domains is achieved, thereby resulting in a bivalent fragment, i.e., a fragment having two antigen-binding sites. Bispecific diabodies are heterodimers of two “crossover” sFv fragments in which the VH and V_Ldomains of the two antibodies are present on different polypeptide chains. Diabodies are described in greater detail in, for example, EP 404,097; WO 93/11161; Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993).

The monoclonal antibodies herein specifically include “chimeric” antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is(are) identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No.4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). “Humanized antibody” is used as a subset of “chimeric antibodies”.

“Humanized” forms of non-human (e .g ., llama or camelid) antibodies are antibodies that contain minimal sequence derived from non-human immunoglobulin. In some embodiments, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from an CDR (hereinafter defined) of the recipient are replaced by residues from an CDR of a non- human species (donor antibody) such as mouse, rat, rabbit, camel, llama, alpaca, or non-human primate having the desired specificity, affinity, and/or capacity. In some instances, framework (“ER”) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies can comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications can be made to further refine antibody performance, such as binding affinity. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin sequence, and all or substantially all of the FR regions are those of a human immunoglobulin sequence, although the FR regions may include one or more individual FR residue substitutions that improve antibody performance, such as binding affinity, isomerization, immunogenicity, etc. The number of these amino acid substitutions in the FR is typically no more than 6 in the H chain, and in the L chain, no more than 3. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, e.g., Jones et al., Nature 321:522-525 (1986); Riechmann et al., Mz/we 332:323-329 (1988); and Presto, Curr. Op. Struct, 670/2:593-596 (1992). See also, for example, Vaswani and Hamilton, Ann. Allergy, Asthmak Immunol. 1 :105-115 (1998); Harris, Biochem. Soc. kansaciions 23-.1035-'WM (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994); and U.S. Pat. Nos. 6,982,321 and 7,087,409.

A “human antibody” is an antibody that possesses an amino-acid sequence corresponding to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art, including phage-display libraries. Hoogenboom and Winter, J. Mol. BioL, 221:36] (1991); Marks et al., J. Mot. BioL, 222:56] (1991). Also available for the preparation of human monoclonal antibodies are methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147(1 ):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, e.g., immunized xenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 regarding XENOMOUSE ™ technology). See also, for example, Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006) regarding human antibodies generated via a human Ilcell hybridoma technology.

The term “hypervariable region,” “HVR,” or “HV,” when used herein refers to the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops. Generally, single-domain antibodies comprise three HVRs (or CDRs): HVR1 (or CDR1), HVR2 (or CDR2), and HVR3 (or CDR3). HVR3 (or CDR3) displays the most diversity of the three HVRs, and is believed to play a unique role in conferring fine specificity to antibodies. See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al., Nature Struct. BioL 3:733-736 (1996).

The term “Complementarity Determining Region” or “CDR” is used to refer to hypervariable regions as defined by the Kabat system. See Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991 ).

A number of HVR delineations are in use and are encompassed herein. The Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia refers instead to the location of the structural loops (Chothia and Lesk, J. Mol. BioL 196:901-917 (1987)). The AbM HVRs represent a compromise between the Kabat HVRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software. The “contact” HVRs are based on an analysis of the available complex crystal structures. The residues from each of these HVRs are noted below in Table I .

H VRs may comprise “extended H VRs” as follows: 24-36 or 24-34 (LI ), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in the V_Land 26-35 (Hl), 50-65 or 49-65 (H2) and 93-102, 94-102, or 95-102 (H3) in the V_H. The variable domain residues are numbered according to Kabat et al., supra, for each of these definitions.

The amino acid residues of a single-domain antibody (such as VHH) are numbered according to the general numbering for VH domains given by Kabat et al. (“Sequence of proteins of immunological interest”, US Public Health Services, NIH Bethesda, Md., Publication No. 91), as applied to VHH domains from Camelids in the article of Riechmann and Muyldermans, J. Immunol. Methods 2000 Jun. 23; 240 (1-2): 185-195. According to this numbering, FR1 of a VHH comprises the amino acid residues at positions 1-30, CDR1 of a VHH comprises the amino acid residues at positions 31-35, FR2 of a VHH comprises the amino acids at positions 36-49, C DR2 of a VHH comprises the amino acid residues at positions 50-65, FR3 of a VHH comprises the amino acid residues at positions 66-94, CDR3 of a VHH comprises the amino acid residues at positions 95-102, and FR4 of a VHH comprises the amino acid residues at positions 103-113. In this respect, it should be noted that — as is well known in the art for V_H domains and for VHH domains — the total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (that is, one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed for by the Kabat numbering).

The expression “variable-domain residue-numbering as in Kabat” or “amino-acid-position numbering as in Kabat,” and variations thereof, refers to the numbering system used for heavy-chain variable domains or light-chain variable domains of the compilation of antibodies in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or HVR of the variable domain. For example, a heavy-chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g. residues 82a, 82b, and 82c, etc. according to Kabat) after heavy-chain FR residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence. Unless indicated otherwise herein, the numbering of the residues in an immunoglobulin heavy chain is that of the EU index as in Kabat et al., supra. The “EU index as in Kaba t” refers to the residue numbering of the human I gGl EU antibody. “Framework” or “ER” residues are those variable-domain residues other than the HVR residues as herein defined. A “human consensus framework” or “acceptor human framework” is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin V_L or V_H framework sequences. Generally, the selection of human immunoglobulin V_Lor V_H sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, 5^lh Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991 ). Examples include for the V_L, the subgroup may be subgroup kappa

I, kappa II, kappa III or kappa IV as in Kabat et al., supra. Additionally, for the VH, the subgroup may be subgroup I, subgroup

II, or subgroup III as in Kabat et al. Alternatively, a human consensus framework can be derived from the above in which particular residues, such as when a human framework residue is selected based on its homology to the donor framework by aligning the donor framework sequence with a collection of various human framework sequences. An acceptor human framework “derived from” a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain pre-existing amino acid sequence changes. In some embodiments, the number of pre-existing amino acid changes are 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less.

An “affinity-matured” antibody is one with one or more alterations in one or more CDRs thereof that result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody that does not possess those alteration(s). In some embodiments, an affinity-matured antibody has nanomolar or even picomolar affinities for the target antigen. Affinity-matured antibodies are produced by procedures known in the art. For example, Marks et al., Bio/TeclinologyVr.119-783 992) describes affinity maturation by V_H- and V_L-domain shuffling. Random mutagenesis of CDR and/or framework residues is described by, for example: Barbas et al. Proc Nat. Acad. Sci. USA 91 :3809-3813 (1994); Schier et al. Gene 169:147-155 (1995); Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al, J. Mol. Biol.226:889--896 (1992).

As used herein, the term “specifically binds,” “specifically recognizes,” or is “specific for” refers to measurable and reproducible interactions such as binding between a target and an antigen binding protein (such as a sdAb), which is determinative of the presence of the target in the presence of a heterogeneous population of molecules including biological molecules. For example, an antigen binding protein (such as a sd Ab) that specifically binds a target (which can be an epitope) is an antigen binding protein (such as a sdAb) that binds this target with greater affinity, avidity, more readily, and/or with greater duration than it binds other targets. In some embodiments, the extent of binding of an antigen binding protein (such as a sdAb) to an unrelated target is less than about 10% of the binding of the antigen binding protein (such as sdAb) to the target as measured, e.g., by a radioimmunoassay (RIA). In some embodiments, an antigen binding protein (such as a sdAb) that specifically binds a target has a dissociation constant (K_d) of <10^-5M,<10^-6M,<10^_7M,<10^_8M,<10^_9M, <10^_|0M, < 10 " M, or <10“¹² M. In some embodiments, an antigen binding protein specifically binds an epitope on a protein that is conserved among the protein from different species. In some embodiments, specific binding can include, but does not require exclusive binding. The term “specificity” refers to selective recognition of on antigen binding protein (such as a sd Ab) for a particular epitope of an antigen. Natural antibodies, for example, are monospecific. The term “multispecific” as used herein denotes that an antigen binding protein has polyepitopic specificity (i.e., is capable of specifically binding to two, three, or more, different epitopes on one biological molecule or is capable of specifically binding to epitopes on two, three, or more, different biological molecules). “Bispecific” as used herein denotes that an antigen binding protein has two different antigen-binding specificities. The term “monospecific” as used herein denotes an antigen binding protein (such as a sd Ab) that has one or more binding sites each of which bind the same epitope of the same antigen.

The term “valent” as used herein denotes the presence of a specified number of binding sites in an antigen binding protein. A natural antibody for example or a full length antibody has two binding sites and is bivalent. As such, the terms “triva lent”, “tetravalent”, “pentavalent” and “hexavalent” denote the presence of two binding site, three binding sites, four binding sites, five binding sites, and six binding sites, respectively, in an antigen binding protein.

“Antibody effector functions” refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity,- Fc receptor binding,- antibody — dependent cell- mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptors); and B cell activation. “Reduced or minimized” antibody effector function means that which is reduced by at least 50% (alternatively 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%) from the wild type or unmodified antibody. The determination of antibody effector function is readily determinable and measurable by one of ordinary skill in the art. In a preferred embodiment, the antibody effector functions of complement binding, complement dependent cytotoxicity and antibody dependent cytotoxicity are affected. In some embodiments, effector function is eliminated through a mutation in the constant region that eliminated glycosylation, e.g., “effector-less mutation.” In one aspect, the effector-less mutation is an N297A or DANA mutation (D265A+N297A) in the C_H2 region. Shields et al.,/. Biol. Chem.llb (9): 6591-6604 (2001). Alternatively, additional mutations resulting in reduced or eliminated effector function include: K322A and L234A/L235A (LALA). Alternatively, effector function can be reduced or eliminated through production techniques, such as expression in host cells that do not glycosylate (e.g ., E. col/jor in which result in an altered glycosylation pattern that is ineffective or less effective at promoting effector function (e.g., Shinkawa et al., /. Biol, Chem.

3466-3473 (2003).

“Antibody-dependent cell-mediated cytotoxicity” or ADCC refers to a form of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils and macrophages) enable these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell with cytotoxins. The antibodies “arm” the cytotoxic cells and are required for killing of the target cell by this mechanism. The primary cells for mediating ADCC, NK cells, express FcyRI II only, whereas monocytes express FcyRI, FcyRII and FcyRI II Fc expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Anno. Bev. Immunol A AS! -32 (1991 ). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. No.5,500,362 or 5,821,337 may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., PNAS 77X495:652-656 (1998). The term “Fc region” herein is used to define a (-terminal region of an immunoglobulin heavy chain, including nativesequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy-chain Fc region is usually defined to stretch from an amino acid residue at position Cy s226 , or from Pro230, to the carboxyl-term inus thereof. The (-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody. Accordingly, a composition of intact antibodies may comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue. Suitable native-sequence Fc regions for use in the antibodies described herein include human IgGl , lgG2 (lgG2 A, lgG2B), lgG3 and lgG4.

“Fc receptor” or “FcR” describes a receptor that binds the Fc region of an antibody. The preferred FcR is a native sequence human FcR. Moreover, a preferred FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the FcyRI, FcyRII, and FcyRIII subclasses, including allelic variants and alternatively spliced forms of these receptors, FcyRII receptors include FcyRI I A (an “activating receptor”) and FcyRI IB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor FcyRI I A contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcyRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain, (see M. Daeron, Annu. Rev. Immunol. 15:203-234 (1997). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol.9: 457-92 (1991); (apel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. tub. Clin.

126:330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term “FcR” herein.

The term “Fc receptor” or “FcR” also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus. Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J. Immunol.24: 249 (1994). Methods of measuring binding to FcRn are known (see, e.g., Ghetie and Ward, Immunol. Today\l: (12): 592-8 (1997); Ghetie et al., Nuture Biotechnology^ (7): 637-40 (1997); Hinton et al., J. Riol. Chern.m (8): 6213-6 (2004); WO 2004/92219 (Hinton et al.). Binding to FcRn in vivo and serum half-life of human FcRn high-affinity binding polypeptides can be assayed, e.g., in transgenic mice or transfected human cell lines expressing human FcRn, or in primates to which the polypeptides having a variant Fc region are administered. WO 2004/42072 (Presto) describes antibody variants which improved or diminished binding to FcRs. See also, e.g., Shields et al., J. Riol. (hem.9(2): 6591-6604 (2001).

“(omplement dependent cytotoxicity” or “(D(” refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (Clq) to antibodies (of the appropriate subclass) which are bound to their cognate antigen. To assess complement activation, a (D( assay, e.g., as described in Gazzano-Santoro et al., J. Immunol. MethodslVl: 163 (1996), may be performed. Antibody variants with altered Fc region amino acid sequences and increased or decreased (Iq binding capability are described in U.S. Pat. No. 6,194,55161 and W099/51642. The contents of those patent publications are specifically incorporated herein by reference. See, also, Idusogie et al. J. Immunol.164: 4178-4184 (2000).

“Binding affinity” generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule [e.g., an antibody) and its binding partner [e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity that reflects a 1 :1 interaction between members of a binding pair. Binding affinity can be indicated by K_d, K_gff, K_on, or K„. The term “K_off”, as used herein, is intended to refer to the off rate constant for dissociation of an antibody (or antigen-binding domain)from the antibody/antigen complex, as determined from a kinetic selection set up, expressed in units of s^_|. The term “K_on”, as used herein, is intended to refer to the on rate constant for association of an antibody (or antigen-binding domain) to the antigen to form the antibody/antigen complex, expressed in units of M^_| s^_|. The term equilibrium dissociation constant “K_g’' or “K_d”, as used herein, refers to the dissociation constant of a particular antibodyantigen interaction, and describes the concentration of antigen required to occupy one half of all of the antibody-binding domains present in a solution of antibody molecules at equilibrium, and is equal to K_off/K_on, expressed in units of M. The measurement of K_d presupposes that all binding agents are in solution. In the case where the antibody is tethered to a cell wall, e.g., in a yeast expression system, the corresponding equilibrium rate constant is expressed as E C50, which gives a good approximation of K_d. The affinity constant, K_a, is the inverse of the dissociation constant, K_d, expressed in units of M^_|.

The dissociation constant (K_D or K_d) is used as an indicator showing affinity of antibodies to antigens. For example, easy analysis is possible by the Scatchard method using antibodies marked with a variety of marker agents, as well as by using BiacoreX (made by Amersham Biosciences), which is an over-the-counter, measuring kit, or similar kit, according to the user's manual and experiment operation method attached with the kit. The K_g value that can be derived using these methods is expressed in units of M (Mol s). An antibody or antigen-binding fragment thereof that specifically binds to a target may have a dissociation constant (K_d) of, for example, <10^_5M, <10^_6M, <10^_7M, <10^{_ 8} M, <10^_9M, <10“¹⁰ M, <10^_l°M, <10-" M, or <10-^|2M.

Binding specificity of the antibody or antigen-binding domain can be determined experimentally by methods known in the art. Such methods comprise, but are not limited to Western blots, ELISA-, RIA-, ECL-, IRMA-, EIA-, Bl Acore-tests and peptide scans.

Half maximal inhibitory concentration (IC₅₀) is a measure of the effectiveness of a substance (such as an antibody) in inhibiting a specific biological or biochemical function. It indicates how much of a particular drug or other substance (inhibitor, such as an antibody) is needed to inhibit a given biological process (e.g., the binding between FAP and B7-1, or component of a process, i.e. an enzyme, cell, cell receptor or microorganism) by half. The values are typically expressed as molar concentration. IC₅₀ is comparable to an EC₅₀ for agonist drug or other substance (such as an antibody). EC₅₀ also represents the plasma concentration required for obtaining 50% of a maximum effect in vivo. As used herein, an “I C₅₀” is used to indicate the effective concentration of an antibody (such as an anti-FAP sd Ab) needed to neutralize 50% of the antigen bioactivity (such as FAP bioactivity) in vitro. IC₅₀or EC₅₀ can be measured by bioassays such as inhibition of ligand binding by FACS analysis (competition binding assay), cell based cytokine release assay, or amplified luminescent proximity homogeneous assay (AlphaLISA).

“Percent (%) amino acid sequence identity” and “homology” with respect to a peptide, polypeptide or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN™ (DN ASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

An “isolated” nucleic acid molecule encoding a construct, antibody, or antigen-binding fragment thereof described herein is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the environment in which it was produced. Preferably, the isolated nucleic acid is free of association with all components associated with the production environment. The isolated nucleic acid molecules encoding the polypeptides and antibodies described herein is in a form other than in the form or setting in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from nucleic acid encoding the polypeptides and antibodies described herein existing naturally in cells. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extra chro mo so mall y or at a chromosomal location that is different from its natural chromosomal location.

The term “radiolabeled,” as in a “radiolabeled” amino acid sequence, radiolabeled” antibody fragment, or “radiolabeled” VHH, refers to the radioisotopic labeling of that amino acid sequence, antibody fragment or VHH, wherein the amino acid sequence, antibody fragment or VHH is labelled by including, coupling, or chemically linking a radionuclide to its amino acid sequence structure.

The terms “radionuclide,” “radioactive nuclide,” “radioisotope,” and “radioactive isotope” are used interchangeably herein and refer to atoms with an unstable nucleus, characterized by excess energy available to be imparted either to a newly created radiation particle within the nucleus or via internal conversion. During this process, the radionuclide is said to undergo radioactive decay, resulting in the emission of gamma ray(s) and/or subatomic particles such as alpha or beta particles. These emissions constitute ionizing radiation. Radionuclides occur naturally, or can be produced artificially. In some embodiments, the radioisotope is both a “y-emitter and P-emitter”, meaning the radioisotope emits both gamma (y) rays and beta (3) particles.

The term “vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”

The term “transfected” or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced" cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein. “Adjuvant setting” refers to a clinical setting in which an individual has had a history of cancer, and generally (but not necessarily) been responsive to therapy, which includes, but is not limited to, surgery (e.g., surgery resection), radiotherapy, and chemotherapy. However, because of their history of cancer, these individuals are considered at risk of development of the disease. Treatment or administration in the “adjuvant setting” refers to a subsequent mode of treatment. The degree of risk (e.g., when an individual in the adjuvant setting is considered as “high risk” or “low risk”) depends upon several factors, most usually the extent of disease when first treated.

“Neoadjuvant setting” refers to a clinical setting in which the method is carried out before the primary/definitive therapy.

The term “pharmaceutical formulation” of “pharmaceutical composition" refers to a preparation that is in such form as to permit the biological activity of the active ingredient to be effective, and that contains no additional components that are unacceptably toxic to a subject to which the formulation would be administered. Such formulations are sterile. A “sterile” formulation is aseptic or free from all living microorganisms and their spores.

It is understood that embodiments of the invention described herein include “consisting” and/or “consisting essentially of” embodiments.

Reference to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X" includes description of “X”.

As used herein, reference to “not” a value or parameter generally means and describes “other than” a value or parameter. For example, the method is not used to treat cancer of type X means the method is used to treat cancer of types other than X.

The term “about X-Y” used herein has the same meaning as “about X to about Y.”

Anti-FAP Constructs

Anti-FAP Single-Domain Antibody Moiety

The isolated anti-FAP construct described herein comprises a single-domain antibody (sdAb) moiety that specifically recognizes FAP (or “anti-FAP sd Ab”). In some embodiments, the isolated anti-FAP construct is an anti-FAP sd Ab.

Single-Domain Antibodies

Exemplary sdAbs include, but are not limited to, heavy chain variable domains from heavy-chain only antibodies (e.g., VHH (variable domain of the heavy chain of the heavy chain antibody) in Camelidae or V_HM (Variable domain of the shark New Antigen Receptor) in cartilaginous fish), binding molecules naturally devoid of light chains, single domains (such as V_H or V_L) derived from conventional 4-chain antibodies, humanized heavy-chain only antibodies, human single-domain antibodies produced by transgenic mice or rats expressing human heavy chain segments, and engineered domains and single domain scaffolds other than those derived from antibodies. The sdAbs may be derived from any species including, but not limited to mouse, rat, human, camel, llama, lamprey, fish, shark, goat, rabbit, and bovine. Single-domain antibodies contemplated herein also include naturally occurring single-domain antibody molecules from species other than Camelidae and sharks.

In some embodiments, the sd Ab is derived from a naturally occurring single-domain antigen binding molecule known as heavy chain antibody devoid of light chains (also referred herein as “heavy chain-only antibodies”, or “ HC Ab ”). Such single domain molecules are disclosed in WO 94/04678 and Hamers-Casterman, C.et al. (1993) Nature 363:446-448, for example. For clarity reasons, the variable domain derived from a heavy chain molecule naturally devoid of light chain is known herein as a VHH to distinguish it from the conventional VH of four chain immunoglobulins. Such a VHH molecule can be derived from antibodies raised in Camelidae species, for example, camel, llama, vicuna, dromedary, alpaca and guanaco. Other species besides Camelidae may produce heavy chain molecules naturally devoid of light chain, and such VH Hs are within the scope of the present application.

In some embodiments, the sd Ab is derived from a variable region of the immunoglobulin found in cartilaginous fish. For example, the sd Ab can be derived from the immunoglobulin isotype known as Novel Antigen Receptor (N AR) found in the serum of shark. Methods of producing single domain molecules derived from a variable region of NAR (“IgNARs”) are described in WO 03/014161 and Streltsov (2005) Protein Sci. 14:2901-2909.

In some embodiments, the sdAb is recombinant, CDR-graf ted, humanized, camelized, de-immunized and/or in vitro generated (e.g, selected by phage display). In some embodiments, the amino acid sequence of the framework regions may be altered by “ca mel ization” of specific amino acid residues in the framework regions. Camelization refers to the replacing or substitution of one or more amino acid residues in the amino acid sequence of a (naturally occurring) VH domain from a conventional 4- chain antibody by one or more of the amino acid residues that occur at the corresponding position(s) in a VHH domain of a heavy chain antibody. This can be performed in a manner known per se, which will be clear to the skilled person, for example on the basis of the further description herein. Such “camelizing” substitutions are preferably inserted at amino acid positions that form and/or are present at the VH-VL interface, and/or at the so-called Camelidae hallmark residues, as defined herein (see for example WO 94/04678, Davies and Riechmann FEES Letters 339: 285-290, 1994; Davies and Riechmann Protein Engineering 9 (6): 531-537, 1996; Riechmann J. Mol. Biol. 259: 957-969, 1996; and Riechmann and Muyldermans J. Immunol. Meth.231: 25-38, 1999).

In some embodiments, the sdAb is a human sdAb produced by transgenic mice or rats expressing human heavy chain segments. See, e.g, US20090307787A1, U.S. Pat. No. 8,754,287, US20150289489A1 , US20100122358A1 , and W02004049794. In some embodiments, the sdAb is affinity matured.

In some embodiments, naturally occurring VHH domains against a particular antigen or target, can be obtained from (naive or immune) libraries of Camelid VHH sequences. Such methods may or may not involve screening such a library using said antigen or target, or at least one part, fragment, antigenic determinant or epitope thereof using one or more screening techniques known per se. Such libraries and techniques are for example described in WO 99/37681, WO 01/90190, WO 03/025020 and WO 03/035694. Alternatively, improved synthetic or semi-synthetic libraries derived from (naive or immune) VHH libraries may be used, such as VHH libraries obtained from [naive or immune) VHH libraries by techniques such as random mutagenesis and/or CDR shuffling, as for example described in WO 00/43507.

In some embodiments, the sd Ab s are generated from conventional four-chain antibodies. See, for example, EP 0368 684, Ward et al. (Nature 1989 Oct. 12; 341 (6242): 544-6), Holt et al. Trends Biotechnol, 2003, 21(11 ):484-490; WO 06/030220; and WO 06/003388.

Because of the unique properties of sdAbs, using VHH domains as single antigen-binding proteins or as antigenbinding domains (i.e. as part of a larger protein or polypeptide) offers a number of significant advantages over the conventional V_H and V_L, scFv and conventional antibody fragments (such as Fab or (Fab')2): I) only a single domain is required to bind an antigen with high affinity, so there is no need to have a second domain, nor to assure that these two domains are present in the correct spatial conformation and configuration (e.g. no need to pair the heavy chain and light chain during folding, no need to use a specially designed linker such as for scFv); 2) VHH domains and other sdAbs can be expressed from a single gene and require no post-translational folding or modifications; 3) VHH domains and other sdAbs can be easily engineered into multivalent and/or multispecific formats (such as those described in the present application); 4) VHH domains and other sdAbs are highly soluble and do not have a tendency to aggregate (as with the mouse-derived “dAbs” described by Ward et al., Nature. 1989 Oct. 12; 341 (6242):544-6); 5) VHH domains and other sdAbs are highly stable against heat, pH, proteases and other denaturing agents or conditions,- 6) VHH domains and other sdAbs are easy and relatively cheap to prepare (even on a large production scale), such as using microbial fermentation, there is no need to use mammalian expression system (required by production of, for example, conventional antibody fragments); 7) VHH domains and other sdAbs are relatively small (approximately 15 kDa, or 10 times smaller than a conventional IgG) compared to conventional 4-chain antibodies and antigenbinding fragments thereof, thus have high(er) tissue penetration ability, such as for solid tumors and other dense tissues,- and 8) VHH domains and other sdAbs can exhibit so-called “cavity-binding properties” (due to their extended CDR3 loop compared to that of conventional V_H domains) and can therefore access targets and epitopes not accessible to conventional 4-chain antibodies and antigen-binding fragments thereof, for example, it has been shown that VHH domains and other sdAbs can inhibit enzymes (see for example WO 1997049805; Transue et al., Proteins. 1998 Sep. 1; 32(4):515-22; Lauwereys et al., EMBO J. 1998 Jul. 1; 17(13):3512-20).

FAP

The amino acid sequence of an exemplary human FAP is SEQ ID NO:77.

Human FAP is a type II transmembrane glycoprotein. It contains a very short cytoplasmic N terminal part [e.g., amino acids 1- 6 of SEQ ID N0:77), a transmembrane region [e.g., amino acids 7-26 of SEQ ID N0:77), and a large extracellular part with an alpha/beta-hydrolase domain and an eight-bladed beta-propeller domain [e.g., amino acids 27-760 of SEQ ID N0:77). A particular human FAP sequence will generally be at least 90% identical SEQ ID NO:77 and contain amino acid residues that identify the amino acid sequence as being human when compared to FAP amino acid sequences of other species (e.g., murine). In some embodiments, a human FAP may be at least about 95%, 96%, 97%, 98%, or 99% identical in amino acid sequence to SEQ ID NO : 77. In some embodiments, a human FAP sequence will display no more than 10 amino acid differences from SEQ ID NO:77. In some embodiments, the human FAP may display no more than 5, 4, 3,2, or 1 amino acid difference SEQ ID N0:77. Percent identity can be determined as described herein. In some embodiments, the anti-FAP sdAb moiety described herein specifically recognizes a FAP polypeptide with 100% amino acid sequence identity to SEQ ID N0:77.

In some embodiments, the anti-FAP sd Ab moiety may cross-react with FAP from species other than human, or other proteins which are structurally related to human FAP (e.g., human FAP homologs). In some embodiments, the anti-FAP sd Ab moiety is completely specific for human FAP and not exhibit species or other types of cross-reactivity. In some embodiments, the anti-FAP sd Ab moiety specifically recognizes a soluble isoform of human FAP.

In some embodiments, the anti-FAP sdAb moiety described herein specifically recognizes the extracellular domain (ECD) of FAP. In some embodiments, the anti-FAP sdAb moiety specifically recognizes the N-terminal portion of the FAP extracellular domain (ECD). In some embodiments, the anti-FAP sdAb moiety specifically recognizes the C-terminal portion of the FAP extracellular domain (ECD). In some embodiments, the anti-FAP sdAb moiety specifically recognizes the middle portion of the FAP extracellular domain (ECD). In some embodiments, the extracellular domain of FAP specifically recognized by the anti-FAP sdAb moiety is at least about 95%, 96%, 97%, 98%, or 99% identical in amino acid sequence to the extracellular domain of human FAP [e.g., amino acids 27-760 of SEQ ID NO:77). In some embodiments, the extracellular domain of FAP specifically recognized by the anti-FAP sdAb moiety is 100% identical in amino acid sequence to the extracellular domain (amino acids 27-760) of the FAP of SEQ ID N 0:77.

Antibody Affinity

Binding specificity of the antibody or antigen-binding domain can be determined experimentally by methods known in the art. Such methods comprise, but are not limited to Western blots, ELISA-, RIA-, ECL-, IRMA-, EIA-, BIAcore-tests and peptide scans.

In some embodiments, the K_d of the binding between the anti-FAP sdAb moiety and FAP is about 10“⁵ M to about

In some embodiments, the IC₅₀of the anti-FAP sdAb moiety is less than 10 nM in an amplified luminescent proximity homogeneous assay (AlphaLISA) with 0.12 nM PD-1 and 0.2 nM FAP. In some embodiments, the IC₅₀ of the onti-FAP sdAb moiety is less than 500 nM in an inhibition of ligand binding by FACS analysis (competition binding assay), or cell based cytokine release assay. In some embodiments, the IC₅₀ of the anti-FAP sdAb moiety is less than 1 nM, about 1 nM to about 10 nM, about 10 nMto about 50 nM, about 50 nM to about 100 nM, about 100 nM to about 200 nM, about 200 nM to about 300 nM, about 300 nM to about 400 nM, or about 400 nM to about 500 nM.

Chimeric or Humanized Antibodies

In some embodiments, the anti-FAP antibody provided herein is a chimeric antibody. Certain chimeric antibodies are described, e.g., in U.S. Pat. No.4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851 -6855 (1984)). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a camelid species, such as llama) and a human constant region. In a further example, a chimeric antibody is a “class switched” antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

In some embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which H VRs, e.g., C D Rs, (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences or are modified to have residues from human antibody sequences. A humanized antibody optionally will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and are further described, e.g., in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. £<£486:10029-10033 (1989); U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing SDR (a-CDR) grafting); Padlan, Mol lmmunol.2t:W)-V)t (1991) (describing “resurfacing”); Dall'Acqua et al., Methods 36:43-60 (2005) (describing “FR shuffling”); Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing the “guided selection” approach to FR shuffling); Vincke et al. J BiolChem.284(5):3273-3284 (2009); and Sulea, I. Humanization of Camelid Single-Domain Antibodies. Methods Mol Biol 2022, 2446,299-312.

Human framework regions that may be used for humanization include but are not limited to: framework regions selected using the “best-fit” method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et a\. Proc. Natl. Acad. Sci. 7/514,89:4285 (1992); and Presta et al. / Immunol, 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619- 1633 (2008)),- and framework regions derived from screening FR libraries (see, e.g., Baca et al., J. Biol. 66^.272:10678-10684 (1997) and Rosok et al., J. Biol Chem.H\ :22611 -22618 (1996)).

In some embodiments, the sdAbs are modified, such as humanized, without diminishing the native affinity of the domain for antigen and while reducing its immunogenicity with respect to a heterologous species. For example, the amino acid residues of the antibody variable domain (VHH) of a llama antibody can be determined, and one or more of the Camelid amino acids, for example, in the framework regions, are replaced by their human counterpart as found in the human consensus sequence, without that polypeptide losing its typical character, i.e. the humanization does not significantly affect the antigen binding capacity of the resulting polypeptide. Humanization of Camelid single-domain antibodies can be obtained by the introduction and mutagenesis of a limited amount of amino acids in a single polypeptide chain. This is in contrast to humanization of scFv, Fab', (Fab')2 and IgG, which requires the introduction of amino acid changes in two chains, the light and the heavy chain and the preservation of the assembly of both chains.

Single-domain antibodies comprising a VHH domain can be humanized to have human like sequences. In some embodiments, the FR regions of the VHH domain used herein comprise at least about any one of 50%, 60%, 70%, 80%, 90%, 95% or more of amino acid sequence homology to human VH framework regions. One exemplary class of humanized VHH domains is characterized in that the VHHs carry an amino acid from the group consisting of glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, tyrosine, tryptophan, methionine, serine, threonine, asparagine, or glutamine at position 45, such as, for example, L45 and a tryptophan at position 103, according to the Kabat numbering. As such, polypeptides belonging to this class show a high amino acid sequence homology to human VH framework regions and said polypeptides might be administered to a human directly without expectation of an unwanted immune response therefrom, and without the burden of further humanization.

Another exemplary class of humanized Camelid single-domain antibodies has been described in WO 03/035694 and contains hydrophobic FR2 residues typically found in conventional antibodies of human origin or from other species, but compensating this loss in hydrophilicity by the charged arginine residue on position 103 that substitutes the conserved tryptophan residue present in V_N from double-chain antibodies. As such, peptides belonging to these two classes show a high amino acid sequence homology to human V_H framework regions, and said peptides might be administered to a human directly without expectation of an unwanted immune response therefrom and without the burden of further humanization.

Exemplary Anti-FAP Constructs and Elements Thereof

In some embodiments, the sdAb moiety comprises a CDR1 comprising the amino acid sequence of any one of SEQ ID NOS:1 -8, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions. In some embodiments, the sdAb moiety comprises a CDR2 comprising the amino acid sequence of any one of SEQ ID NOS:9-16, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions. In some embodiments, the sdAb moiety comprises a CDR3 comprising the amino acid sequence of any one of SEQ ID NOS:17-24, or a variant thereof comprising up to about 3 (such as about any of 1 , 2, or 3) amino acid substitutions.

In some embodiments, the sdAb moiety comprises: a CDR1 comprising the amino acid sequence of any one of SEQ ID NOS:1 -8, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; a CDR2 comprising the amino acid sequence of any one of SEQ ID NOS:9-16, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; and/or a CDR3 comprising the amino acid sequence of any one of SEQ ID N OS: 17-24, or a variant thereof comprising up to about 3 (such as about any of 1 , 2, or 3) amino acid substitutions.

In some embodiments, the sdAb moiety comprises: a CDR1 comprising the amino acid sequence of any one of SEQ ID NOS:1 -8, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions,- a CDR2 comprising the amino acid sequence of any one of SEQ ID NOS:9-16, or a variant thereof comprising up to about 3 (such as about any of 1,2, or 3) amino acid substitutions; and a CDR3 comprising the amino acid sequence of any one of SEQ ID NOS:17- 24, or a variant thereof comprising up to about 3 (such as about any of 1 , 2, or 3) amino acid substitutions.

In some embodiments, the sd Ab moiety comprises: a CDR1 comprising the amino acid sequence of any one of SEQ ID NOS:1 -8; a CDR2 comprising the amino acid sequence of any one of SEQ ID NOS:9-16; and/or a CDR3 comprising the amino acid sequence of any one of SEQ ID NOS:17-24.

In some embodiments, the sd Ab moiety comprises: a CDR1 comprising the amino acid sequence of any one of SEQ ID NOS:1 -8; a CDR2 comprising the amino acid sequence of any one of SEQ ID NOS:9-16; and a CDR3 comprising the amino acid sequence of any one of SEQ ID NOS:17-24.

In some embodiments, the sdAb moiety comprises: a CDR1 comprising the amino acid sequence of SEQ ID N0:l or a variant thereof comprising up to about 3 (such as about any of 1 , 2, or 3) amino acid substitutions, a C DR2 comprising the amino acid sequence of SEQ ID NO:9 or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions, and a CDR3 comprising the amino acid sequence of SEQ ID N 0:17 or a variant thereof comprising up to about 3 (such as about any of 1,2, or 3) amino acid substitutions. In some embodiments, the sdAb moiety comprises: a CDR1 comprising the amino acid sequence of SEQ ID N0:2 or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions, a CDR2 comprising the amino acid sequence of SEQ ID N0:l 0 or a variant thereof comprising up to about 3 (such as about any of 1,2, or 3) amino acid substitutions, and a CDR3 comprising the amino acid sequence of SEQ ID N0:l 8 or a variant thereof comprising up to about 3 (such as about any of 1 , 2, or 3) amino acid substitutions. In some embodiments, the sdAb moiety comprises: a CDR1 comprising the amino acid sequence of SEQ ID N0:3 or a variant thereof comprising up to about 3 (such as about any of 1,2, or 3) amino acid substitutions, a CDR2 comprising the amino acid sequence of SEQ ID N0:l 1 or a variant thereof comprising up to about 3 (such as about any of 1,2, or 3) amino acid substitutions, and a CDR3 comprising the amino acid sequence of SEQ ID N0:19 or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions. In some embodiments, the sdAb moiety comprises: a CDR1 comprising the amino acid sequence of SEQ ID N0:4 or a variant thereof comprising up to about 3 (such as about any of 1,2, or 3) amino acid substitutions, a CDR2 comprising the amino acid sequence of SEQ ID N0:l 2 or a variant thereof comprising up to about 3 (such as about any of 1,2, or 3) amino acid substitutions, and a CDR3 comprising the amino acid sequence of SEQ ID N 0:20 or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions. In some embodiments, the sdAb moiety comprises: a CDR1 comprising the amino acid sequence of SEQ ID N0:5 or a variant thereof comprising up to about 3 (such as about any of 1,2, or 3) amino acid substitutions, a CDR2 comprising the amino acid sequence of SEQ ID N0:l 3 or a variant thereof comprising up to about 3 (such as about any of 1,2, or 3) amino acid substitutions, and a CDR3 comprising the amino acid sequence of SEQ ID N0:21 or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions. In some embodiments, the sdAb moiety comprises: a CDR1 comprising the amino acid sequence of SEQ ID N0:6 or a variant thereof comprising up to about 3 (such as about any of 1 , 2, or 3) amino acid substitutions, a CDR2 comprising the amino acid sequence of SEQ ID N0:l 4 or a variant thereof comprising up to about 3 (such as about any of 1,2, or 3) amino acid substitutions, and a CDR3 comprising the amino acid sequence of SEQ ID N 0:22 or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions. In some embodiments, the sdAb moiety comprises: a CDR1 comprising the amino acid sequence of SEQ ID N 0 :7 or a variant thereof comprising up to about 3 (such as about any of 1 , 2, or 3) amino acid substitutions, a CDR2 comprising the amino acid sequence of SEQ ID N0:l 5 or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions, and a CDR3 comprising the amino acid sequence of SEQ ID NO:23 or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions. In some embodiments, the sdAb moiety comprises: a C DR1 comprising the amino acid sequence of SEQ ID N 0:8 or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions, a CDR2 comprising the amino acid sequence of SEQ ID N0:l 6 or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions, and a C DR3 comprising the amino acid sequence of SEQ ID N0:24 or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions.

In some embodiments, the sdAb moiety comprises: a CDR1 comprising the amino acid sequence of SEQ ID N0:l , a CDR2 comprising the amino acid sequence of SEQ ID N0:9, and a CDR3 comprising the amino acid sequence of SEQ ID N0:l 7. In some embodiments, the sdAb moiety comprises: a CDR1 comprising the amino acid sequence of SEQ ID N0:2, a CDR2 comprising the amino acid sequence of SEQ ID N0:10, and a CDR3 comprising the amino acid sequence of SEQ ID N0:18. In some embodiments, the sdAb moiety comprises: a CDR1 comprising the amino acid sequence of SEQ ID N 0:3, a CDR2 comprising the amino acid sequence of SEQ ID NO :11 , and a CDR3 comprising the amino acid sequence of SEQ ID N 0:19. In some embodiments, the sdAb moiety comprises: a CDR1 comprising the amino acid sequence of SEQ ID N0:4, a CDR2 comprising the amino acid sequence of SEQ ID N0:12, and a CDR3 comprising the amino acid sequence of SEQ ID N0:20. In some embodiments, the sdAb moiety comprises: a CDR1 comprising the amino acid sequence of SEQ ID N 0:5, a CDR2 comprising the amino acid sequence of SEQ ID N0:l 3, and a CDR3 comprising the amino acid sequence of SEQ ID N0:21 . In some embodiments, the sdAb moiety comprises: a CDR1 comprising the amino acid sequence of SEQ ID N0:6, a CDR2 comprising the amino acid sequence of SEQ ID N 0:14, and a C DR3 comprising the amino acid sequence of SEQ ID N 0:22. In some embodiments, the sdAb moiety comprises: a CDR1 comprising the amino acid sequence of SEQ ID N0:7, a CDR2 comprising the amino acid sequence of SEQ ID NO:15, and a CDR3 comprising the amino acid sequence of SEQ ID N0:23. In some embodiments, the sdAb moiety comprises: a CDR1 comprising the amino acid sequence of SEQ ID N0:8, a CDR2 comprising the amino acid sequence of SEQ ID NO:16, and a CDR3 comprising the amino acid sequence of SEQ ID NO :24.

In some versions, the sdAb moiety comprises a VHH domain comprising the amino acid sequence of SEQ ID NO :57 or a variant thereof having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID N 0:57, wherein the CDR1 comprises the amino acid sequence of SEQ ID N0:l, the CDR2 comprises the amino acid sequence of SEQ ID NO:9, and the CDR3 comprises the amino acid sequence of SEQ ID NO :17. In some versions, the sdAb moiety comprises a VHH domain comprising the amino acid sequence of SEQ ID N 0:59 or a variant thereof having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO -.59 , wherein the CDR 1 comprises the amino acid sequence of SEQ ID N0:2, the CDR2 comprises the amino acid sequence of SEQ ID N0:l 0, and the CDR3 comprises the amino acid sequence of SEQ ID NO:18. In some versions, the sdAb moiety comprises a VHH domain comprising the amino acid sequence of SEQ ID NO :61 or a variant thereof having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID N0:61 , wherein the CDR1 comprises the amino acid sequence of SEQ ID N0:3, the CDR2 comprises the amino acid sequence of SEQ ID N0:l 1, and the CDR3 comprises the amino acid sequence of SEQ ID N 0:19. In some versions, the sdAb moiety comprises a VHH domain comprising the amino acid sequence of SEQ ID NO :63 or a variant thereof having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID N0:63, wherein the CDR1 comprises the amino acid sequence of SEQ ID N 0:4, the C DR2 comprises the amino acid sequence of SEQ ID N 0 :12, and the CDR3 comprises the amino acid sequence of SEQ ID N0:20. In some versions, the sdAb moiety comprises a VHH domain comprising the amino acid sequence of SEQ ID N 0:65 or a variant thereof having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO :65, wherein the CDR 1 comprises the amino acid sequence of SEQ ID N 0:5, the CDR2 comprises the amino acid sequence of SEQ ID N0:l 3, and the CDR3 comprises the amino acid sequence of SEQ ID N0:21. In some versions, the sdAb moiety comprises a VHH domain comprising the amino acid sequence of SEQ ID NO :67 or a variant thereof having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID N 0:67, wherein the CDR1 comprises the amino acid sequence of SEQ ID NO:6, the CDR2 comprises the amino acid sequence of SEQ ID NO:14, and the CDR3 comprises the amino acid sequence of SEQ ID N 0:22. In some versions, the sd Ab moiety comprises a VHH domain comprising the amino acid sequence of SEQ ID NO :69 or a variant thereof having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID N0:69, wherein the CDR1 comprises the amino acid sequence of SEQ ID NO:7, the CDR2 comprises the amino acid sequence of SEQ I D N0:l 5, and the CDR3 comprises the amino acid sequence of SEQ ID N0:23. In some versions, the sd Ab moiety comprises a VHH domain comprising the amino acid sequence of SEQ ID N 0:71 or a variant thereof having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:71 , wherein the CDR1 comprises the amino acid sequence of SEQ ID NO:8, the CDR2 comprises the amino acid sequence of SEQ ID N0:l 6, and the CDR3 comprises the amino acid sequence of SEQ ID NO:24.

The sequences of the CDRs noted herein are provided in Table 3.

The anti-FAP sdAb moiety may comprise one or more “hallmark residues” in one or more of the ER sequences. In some embodiments, the anti-FAP sdAb moiety may comprise a VHH domain comprising the amino acid sequence of any one of the following: a-1 ) the amino acid residue at position 37 is selected from the group consisting of F, Y, L, I, and V (such as Y or such as F); a-2) the amino acid residue at position 44 is selected from the group consisting of A, G, E, D, G, Q, R, S, and L (such as G, E, or Q); a-3) the amino acid residue at position 45 is selected from the group consisting of L, R and C (such as L or R); a- 4) the amino acid residue at position 103 is selected from the group consisting of G, W, R and S (such as W or R, or such as W); and a-5) the amino acid residue at position 108 is Q; or b-1) the amino acid residue at position 37 is selected from the group consisting of F, Y, L, I, and V (such as Y or such as F); b-2) the amino acid residue at position 44 is selected from the group consisting of E and Q; b-3) the amino acid residue at position 45 is R; b-4) the amino acid residue at position 103 is selected from the group consisting of G, W, R and S (such as W); and b-5) the amino acid residue at position 108 is selected from the group consisting of Q and L (such as Q); wherein the amino acid position is according to Rabat numbering. It should be noted that these “hallmark residues” at amino acid positions 37, 44, 45, 103 and 108 according to Rabat numbering apply to anti- FAP sdAb moieties of natural VHH sequences, and can be substituted during humanization. For example, Q at amino acid position 108 according to Rabat numbering can be optionally humanized to L. Other humanized substitutions will be clear to those skilled in the art. For example, potentially useful humanizing substitutions can be determined by comparing the FR sequences of a naturally occurring VHH with the corresponding FR sequences of one or more closely related human V_H, then introducing one or more of such potentially useful humanizing substitutions into said VHH using methods known in the art (also as described herein). The resulting humanized VHH sequences can be tested for their FAP binding affinity, for stability, for ease and level of expression, and/or for other desired properties. Possible residue substitutions may also come from an antibody V_H domain wherein the VH/VL interface comprises one or more highly charged amino acid residues. The anti-FAP sdAb moiety described herein can be partially or fully humanized.

In some embodiments, there is provided an anti-FAP sdAb moiety comprising a VHH domain comprising the amino acid sequence of any one of SEQ ID N 0 s:57, 59, 61, 63, 65, 67, 69, and 71 , or a variant thereof having at least about 80% (such as at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identify to any one of SEQ ID NOs: 57, 59, 61, 63, 65, 67, 69, and 71. In some embodiments, there is provided an anti-FAP sdAb moiety comprising a VHH domain comprising the amino acid sequence of any one of SEQ ID NOs: 57, 59, 61, 63, 65, 67, 69, and 71, or a variant thereof comprising up to about 3 (such as about any of 1,2, or 3) amino acid substitutions in the VHH domain. In some embodiments, the anti-FAP sd Ab moiety comprising the VHH domain comprising the amino acid sequence of any one of SEQ ID NOs: 57, 59, 61, 63, 65, 67, 69, and 71 or the variant thereof comprises amino acid substitutions in CDRs, such as the CDR1 , and/or the CDR2, and/or the CDR3 of any one of SEQ ID NOs: 57,59, 61,63,65, 67,69, and 71. In some embodiments, the anti- FAP sd Ab moiety comprising the VHH domain comprising the amino acid sequence of any one of SEQ ID NOs: 57, 59, 61,63, 65, 67,69, and 71 or the variant thereof comprises the CDR1, CDR2, and CDR3 of anyone of SEQ ID NOs: 57, 59, 61,63, 65,67, 69, and 71 , and the amino acid substitutions are in F Rs, such as the FR 1 , and/or the FR2, and/or the FR3, and/or the FR4 of any one of SEQ ID NOs: 57, 59, 61, 63, 65, 67, 69, and 71.

Constructs Comprising the Anti-FAP sdAb Moiety

The anti-FAP construct comprising the anti-FAP sd Ab moiety can be of any possible format.

In some embodiments, the anti-FAP construct comprising the anti-FAP sd Ab moiety may further comprise additional polypeptide sequences, such as one or more antibody moieties, or Fc fragment of immunoglobulin. Such additional polypeptide sequences may or may not change or otherwise influence the (biological) properties of the sd Ab, and may or may not add further functionality to the sdAb described herein. In some embodiments, the additional polypeptide sequences confer one or more desired properties or functionalities to the sdAb of the present invention. In some embodiments, the anti-FAP construct is a chimeric antigen receptor (CAR) comprising an extracellular antigen binding domain comprising one or more anti-FAP sdAb moiety described herein.

In some embodiments, the additional polypeptide sequences may be a second antibody moiety (such as sdAb, scFv, full-length antibody) that specifically recognizes a second antigen. In some embodiments, the second antigen is not FAP. In some embodiments, the second antibody moiety specifically recognizes the same epitope on FAP as the anti-FAP sdAb described herein. In some embodiments, the second antibody moiety specifically recognizes a different epitope on FAP as the anti-FAP sdAb described herein.

In some embodiments, the additional polypeptide sequences may increase the antibody construct half-life, solubility, or absorption, reduce immunogenicity or toxicity, eliminate or attenuate undesirable side effects, and/or confer other advantageous properties to and/or reduce undesired properties of the anti-FAP construct of the invention, compared to the anti-FAP sdAb described herein per se. Some non-limiting examples of such additional polypeptide sequences are serum proteins, such as human serum albumin (see for example WO 00/27435) or haptenic molecules (for example haptens that are recognized by circulating antibodies, see for example WO 98/22141). It was shown that linking fragments of immunoglobulins (such as V_H domains) to serum albumin or fragments thereof may increase antibody half-life (see e.g. WO 00/27435 and WO 01/077137). Thus, in some embodiments, the anti-FAP construct of the present invention may comprise an anti-FAP sdAb moiety described herein linked to serum albumin (or to a suitable fragment thereof), optionally via a suitable linker (such as peptide linker). In some embodiments, the anti-FAP sdAb moiety described herein can be linked to a fragment of serum albumin at least comprising serum albumin domain III. (see PCT/EP2007/002817).

Heavy Chain-Only Antibody (HCAb)

In some embodiments, anti-FAP sdAb moiety described herein can be linked to one or more [preferably human) C_H2 and/or C_H3 domains, optionally via a linker sequence, to increase its half-life in vivo. Thus in some embodiments, the anti-FAP construct is an HCAb (hereinafter referred to as “anti-FAP HCAb”) comprising an anti-FAP sdAb moiety described herein fused to an Fc fragment of an immunoglobulin, such as IgA, IgD, IgE, IgG, and I gM. In some embodiments, the anti-FAP HCAb comprises an Fc sequence of IgG, such as any of I gG 1 , 1 gG2, 1 g G3, or Ig G4. In some embodiments, the Fc fragment is a human Fc. In some embodiments, the Fc fragment is a human IgGl Fc. In some embodiments, the anti-FAP HCAb is monomeric. In some embodiments, the anti-FAP HCAb is dimeric. In some embodiments, the anti-FAP sd Ab moiety and the Fc fragment are optionally connected by a peptide linker. In some embodiments, the peptide linker is a peptide linker disclosed herein. In some embodiments, the peptide linker is a mutated human IgGl hinge (see SEQ ID NO:445 of US 11,673,954, which is incorporated herein by reference). In some embodiments, the peptide linker comprises the amino acid sequence of

(SEQ ID NO:78). In some embodiments, the peptide linker comprises the amino acid sequence of

(SEQ ID N 0 :79). In some embodiments, the peptide linker comprises the amino acid sequence of GPGGP (SEQ ID N0:80). Other suitable peptide linkers are provided in Klein JS, Jiang S, Galimidi RP, Keeffe JR, B jorkman PJ. Design and characterization of structured protein linkers with differing flexibilities. Protein Eng Des Sei. 2014 0ct;27(10):325- 30.

Thus in some embodiments, there is provided an anti-FAP HCAb comprising an sdAb moiety of the invention fused to an Fc fragment of an immunoglobulin. In some embodiments, the Fc fragment is a human IgGl Fc. An exemplary IgGl Fc is:

In some embodiments, the anti-FAP HCAb is monomeric. In some embodiments, the anti-FAP HCAb is dimeric. In some embodiments, the anti-FAP sdAb moiety and the Fc fragment are optionally connected by a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID N 0:78, SEQ ID NO :79, or SEQ ID NO :80. In some embodiments, the anti-FAP sdAb moiety is camelid, chimeric, human, partially humanized, or fully humanized.

In some embodiments, there is provided an anti-FAP HCAb comprising the amino acid sequence of SEQ ID NO:75, or a variant at least 80%, 85%, 90%, 95%, 99% identical thereto.

Multivalent and/or Multispecific Antibodies

In some embodiments, the anti-FAP construct comprises an anti-FAP sdAb moiety described herein fused to one or more other antibody moiety (such as an antibody moiety that specifically recognizes FAP or another antigen). The one or more other antibody moiety can be of any antibody or antibody fragment format, such as a multispecific sdAb (such as bispecific sdAb), a full-length antibody, a Fab, a Fab', a (Fab')2, an Fv, a single chain Fv (scFv), an scFv-scFv, a minibody, a diabody, or a sdAb. For a review of certain antibody fragments, see Hudson et al. Nat. Med. 9:129-134(2003). For a review of scFv fragments, see, e.g., Pluckthiin, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For discussion of Fab and F(ab')₂ fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Pat. No. 5,869,046. For a review of multispecific antibodies, see Weidle et al., Cancer Genomics Proteomics, 10(1 ):1 -18, 2013; Geering and Fussenegge r, Trends B iotechnal ., 33(2):65 - 79, 2015; Stamova et al., Antibodies, 1 (2): 172-198, 2012. Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161; Hudson et al., Nat. Med.9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA%: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med.9:129-134 (2003). Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g. E. co/ior phage), as described herein. In some embodiments, the one or more other antibody moiety is antibody mimetics, which are small engineered proteins comprising antigen-binding domains reminiscent of antibodies (Geering and Fussenegger, Trends B io technol., 33(2):65-79, 2015). These molecules are derived from existing human scaffold proteins and comprise a single polypeptide. Exemplary antibody mimetics that can be comprised within the anti-FAP construct described herein can be, but are not limited to, a designed ankyrin repeat protein (D ARPin,- comprising 3-5 fully synthetic ankyrin repeats flanked by N- and C-terminal Cap domains), an avidity multimer (avimer; a high-affinity protein comprising multiple A domains, each domain with low affinity for a target), or an Anticalin (based on the scaffold of lipocalins, with four accessible loops, the sequence of each can be randomized).

Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature WS.537 (1983)), WO 93/08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), and “knob-in-hole” engineering (see, e.g., U.S. Pat. No. 5,731,168). Multi-specific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc- heterodimeric molecules (WO 2009/089004A1); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science, TIE): 81 (1985)); using leucine zippers to produce bi-specific antibodies (see, e.g., Kostelny et al., J. Immuno/., 148(5):1547-1553 (1992)); using “diabody” technology for making bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol, \52-5338 (1994)); and preparing trispecific antibodies as described, e.g., in Tutt et al. J. Immunol. W-. 60 (1991); and creating polypeptides comprising tandem single-domain antibodies (see, e.g., U.S. Patent Application No. 20110028695; and Conrath et al. J. Biol. Chem., 2001; 276(10):7346-50). Engineered antibodies with three or more functional antigen binding sites, including “Octopus antibodies,” are also included herein (see, e.g., US 2006/0025576A1 ).

In some embodiments, the anti-FAP construct comprises a first anti-FAP sdAb moiety of the invention described herein fused to a second anti-FAP sdAb moiety of the invention. The first and second anti-FAP sdAb moieties can include any anti-FAP sdAb moieties described herein. The first and second anti-FAP sdAb moieties can be fused via a peptide linker.

In some embodiments, there is provided an anti-FAP construct comprising the amino acid sequence of SEQ ID N 0 :73, or a variant at least 80%, 85%, 90%, 95%, 99% identical thereto.

Peptide Linkers

In some embodiments, the two or more antibody moieties within the anti-FAP construct can be optionally connected by a peptide linker. The length, the degree of flexibility and/or other properties of the peptide linker(s) used in the anti-FAP construct may have some influence on properties, including but not limited to the affinity, specificity or avidity for one or more particular antigens or epitopes. For example, longer peptide linkers may be selected to ensure that two ad jacen t domains do not sterically interfere with one another. In some embodiment, a peptide linker comprises flexible residues (such as glycine and serine) so that the adjacent domains are free to move relative to each other. For example, a glycine-serine doublet can be a suitable peptide linker.

The peptide linker can be of any suitable length. In some embodiments, the peptide linker is at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100 or more amino acids long. In some embodiments, the peptide linker is no more than about any of 100, 75,50, 40, 35, 30,25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or fewer amino acids long. In some embodiments, the length of the peptide linker is any of about 1 amino acid to about 10 amino acids, about 1 amino acid to about 20 amino acids, about 1 amino acid to about 30 amino acids, about 5 amino acids to about 15 amino acids, about 10 amino acids to about 25 amino acids, about 5 amino acids to about 30 amino acids, about 10 amino acids to about 30 amino acids long, about 30 amino acids to about 50 amino acids, about 50 amino acids to about 100 amino acids, or about 1 amino acid to about 100 amino acids.

The peptide linker may have a naturally occurring sequence, or a non-naturally occurring sequence. For example, a sequence derived from the hinge region of heavy chain only antibodies may be used as the linker. See, for example, WO1996/34103. In some embodiments, the peptide linker is a mutated human IgGl hinge (see SEQ ID N0:445 of US 11,673,954). In some embodiments, the peptide linker is a flexible linker. Exemplary flexible linkers include glycine polymers (GJ,, glycineserine polymers (including, for example, (GS)_n, (GSGGS (SEQ ID NO:82))_n, (GGGS (SEQ ID N0:83))„, and (GGGGS (SEQ ID NO:84))_n, where n is an integer of at least one, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20,25 or more, and/or, optionally, up to 10, 15,20, 25, 30, 35, or more), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. In some embodiments, the peptide linker comprises the amino acid sequence of GGGGSGGGS (SEQ ID NO :78). In some embodiments, the peptide linker comprises the amino acid sequence of GGGGSGGGGSGGGGS (SEQ ID N 0 :79). In some embodiments, the peptide linker comprises the amino acid sequence of GPGGP (SEQ ID N0:80). In some embodiments, the peptide linker comprises the amino acid sequence of

(SEQ ID N0:85). In some embodiments, the peptide linker comprises the amino acid sequence of EPKSSDKTHTSPPSP (SEQ ID N 0:86). Other suitable peptide linkers are provided in Klein JS, Jiang S, Galimidi RP, Keeffe JR, B jorkman PJ. Design and characterization of structured protein linkers with differing flexibilities. Protein Eng Des Sei. 2014 0ct;27(10):325-30.

In some embodiments, the anti-FAP construct comprising an anti-FAP sdAb moiety and one or more other antibody moiety is monospecific. In some embodiments, the anti-FAP construct comprising an anti-FAP sdAb moiety and one or more other antibody moiety is multispecific (such as bispecific). Multispecific molecules are molecules that have binding specificities for at least two different antigens or epitopes (e.g., bispecific antibodies have binding specificities for two antigens or epitopes). Multispecific molecules with more than two valencies and/or specificities are also contemplated. For example, trispecific antibodies can be prepared. Tutt et al. J. Immunol. WI: 60 (1991). It is to be appreciated that one of skill in the art could select appropriate features of individual multispecific molecules described herein to combine with one another to form a multi-specific anti-FAP molecule of the invention.

In some embodiments, the anti-FAP construct is multivalent but monospecific, i.e., the anti-FAP construct comprises an anti-FAP sdAb moiety described herein and at least a second antibody moiety specifically recognizing the same FAP epitope as the anti-FAP sdAb moiety. In some embodiments, the one or more antibody moiety specifically recognizing the same FAP epitope as the anti-FAP sdAb moiety described herein may comprise the same C DRs and/or the same VHH amino acid sequence as the anti-FAP sdAb moiety. For example, the anti-FAP construct may comprise two or more anti-FAP sdAb moieties described herein, wherein the two or more onti-FAP sdAb moieties ore the some. In some embodiments, the onti-FAP sdAb moieties ore optionally connected by peptide linker(s).

In some embodiments, the anti-FAP construct is multivalent and multispecific, i.e., the anti-FAP construct comprises an anti-FAP sdAb moiety described herein and at least a second antibody moiety specifically recognizing a second antigen other than FAP, or a different FAP epitope recognized by the anti-FAP sdAb moiety. In some embodiments, the second antibody moiety is a sdAb. In some embodiments, the second antibody moiety specifically recognizes human serum albumin (USA). In some embodiments, the sdAb moiety specifically recognizing FAP is N terminal or C terminal to the second antibody moiety. In some embodiments, the anti-FAP construct is trivalent and bispecific. In some embodiments, the anti-FAP construct comprises two anti-FAP sdAbs described herein and a second antibody moiety (such as an anti-HSA sdAb), wherein the second antibody moiety is in between the two anti-FAP sdAbs. In some embodiments, the antibody moieties are optionally connected by peptide linker(s).

The monospecific or multispecific anti-FAP construct comprising two or more sdAb moieties specifically recognizing FAP may have increase avidity compared to that of a single anti-FAP sdAb moiety described here.

Bispecific Antibodies Comprising sdAb Fused to Full-Length Antibody

In some embodiments, the anti-FAP construct comprises an anti-FAP sdAb moiety described herein fused to a second antibody moiety, wherein the second antibody moiety is a full-length antibody (such as anti-TIGIT full-length antibody). The construct comprising bi-specificity against FAP and TIGIT will be hereinafter referred to as “anti-FAP/TI GIT antibody”, “anti- FAP/TI GIT construct”, or “FAPXTIGIT antibody”.

In some embodiments, the anti-FAP construct comprises an anti-FAP sdAb moiety described herein fused to a second antibody moiety, wherein the second antibody moiety is a full-length antibody (such as anti-TIM-3 full-length antibody). The construct comprising bi-specificity against FAP and TIM-3 will be hereinafter referred to as “anti-FAP/TIM-3 antibody”, “anti- FAP/TIM-3 construct”, or “FAPXTIM-3 antibody”.

In some embodiments, the anti-FAP construct comprises an anti-FAP sdAb moiety described herein fused to a second antibody moiety, wherein the second antibody moiety is a full-length antibody (such as anti-LAG-3 full-length antibody). The construct comprising bi-specificity against FAP and LAG-3 will be hereinafter referred to as “an ti-FAP/LAG-3 antibody”, “anti- FAP/LAG-3 construct”, or “F AP X LAG- 3 antibody”.

TIGIT, TIM-3 and LAG-3 are inhibitory immune checkpoint molecules.

Anti-FAP Antibody Variants

In some embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleic acid sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding. a) Substitution, Insertion, Deletion and Variants

In some embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the HVRs and FRs. Conservative substitutions are shown in Table 2 under the heading of “Preferred substitutions.” More substantial changes are provided in Table 2 under the heading of “exemplary substitutions,” and as further described below in reference to amino acid side chain classes. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.

(6) Aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further study will have modifications (e.g ., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and/or will have substantially retained certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, e.g., using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies displayed on phage and screened for a particular biological activity (e.g. binding affinity).

Alterations (e.g., substitutions) may be made in HVRs, e.g., to improve antibody affinity. Such alterations may be made in HVR “hotspots,” i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol.lMl-VN-Vtb (2008)), and/or SDRs (a-CDRs), with the resulting variant VH or VL being tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries has been described, e.g., in Hoogenboom et al. in Methods in Molecular Biology ]lbt\ -11 (O'Brien et al., ed., Human Press, Totowa, N.J., (2001)) In some embodiments of affinity maturation, diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonudeo tide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method to introduce diversity involves HVR-directed approaches, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.

In some embodiments, substitutions, insertions, or deletions may occur within one or more HVRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs. Such alterations may be outside of HVR “hotspots” or CDRs. In some embodiments of the variant VHH sequences provided above, each HVR either is unaltered, or contains no more than one, two or three amino acid substitutions.

A useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called “alanine scanning mutagenesis” as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with antigen is affected. Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions. Alternatively, or additionally, a crystal structure of an antigen-antibody complex to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide which increases the serum half-life of the antibody. b) Glycosylation Variants

In some embodiments, an anti-FAP construct provided herein is altered to increase or decrease the extent to which the construct is glycosylated. Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.

Where the anti-FAP construct comprises an Fc region, the carbohydrate attached thereto may be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al. TIBTECH}5-2b-32 (1997). The oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the “stem” of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharide in an anti-FAP construct of the present application may be made in order to create antibody variants with certain improved properties.

In some embodiments, antibody variants are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such antibody may be from 1 % to 80%, from 1 % to 65%, from 5% to 65% or from 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e.g ., complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (EU numbering of Fc region residues); however, Asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e ., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to “defucosylated” or “fucose-deficient” antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; W02005/053742; W02002/031140; Okazaki et al.7. Mol. 6to/.336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng.il-. 614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Led 3 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys.2V):522-5$5 (1986); US Patent Application No. US 2003/0157108 Al, Presta, L; and WO 2004/056312 Al, Adams et al., especially at Example II), and knockout cell lines, such as alpha-1, 6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng.il-. 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680 -688 (2006); and W02003/085107).

Anti-FAP construct variants are further provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, e.g., in WO 2003/011878 (Jean- Mairet et al.),- U.S. Pat. No.6,602,684 (Umana et al.),- and US 2005/0123546 (Umana et al.). Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.). c) Fc Region Variants

In some embodiments, one or more amino acid modifications may be introduced into the Fc region of the anti-FAP construct provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgGl, I gG2, lgG3 or Ig G4 Fc region) comprising an amino acid modification (e.g. a substitution) at one or more amino acid positions.

In some embodiments, the present application contemplates an anti-FAP construct variant that possesses some but not all effector functions, which make it a desirable candidate for applications in which the half-life of the anti-FAP construct in vivo is important yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks Fey R binding (hence likely lacking ADCC activity), but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express Fey Rl 11 only, whereas monocytes express FcyRI, Fey RII and Fey RII I FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol.') :457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Pat. No. 5,500,362 (see, e.g. Hellstrom, I. et al. Proc. Hat'l Acad. Sci. USAMJtffl- 7063 (1986)) and Hellstrom, I et al., Proc. Hat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al.,/. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays methods maybe employed (see, for example, ACTI™ non-radioactive cytotoxicity assay for flow cytometry (Cell Technology, Inc. Mountain View, Calif.; and CytoTox 96® nonradioactive cytotoxicity assay (Promega, Madison, Wis.). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Hat'l Acad. Sci. ZZ£495:652-656 (1998). Clq binding assays may also be carried out to confirm that the antibody is unable to bind Clq and hence lacks CDC activity. See, e.g., Clq and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To assess complement activation, a CDC assay may be performed (see, for example, Gazzano-Santoro et al., /. Immunol. Methods 202;] (A (1996); Cragg, M. S. et al., Blood} 01 :1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, 67^/103:2738-2743 (2004)). FcRn binding and in vivo dearance/ha lf-1 ife determinations can also be performed using methods known in the art (see, e.g ., Petkova, S. B. et al., Intl'. Immunol. 18(12):1759-1769 (2006)).

Antibodies with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Pat. No.6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine (U.S. Pat. No. 7,332,581).

Certain antibody variants with improved or diminished binding to FcRs are described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, and Shields et al., /. Biol. Chem.9(2): 6591-6604 (2001)). In some embodiments, on anti-FAP construct variant comprises an Fc region with one or more amino acid substitutions which improve ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the Fc region (EU numbering of residues).

In some embodiments, alterations are made in the Fc region that result in altered (i.e., either improved or diminished) Clq binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164 : 4178-4184 (2000).

In some embodiments, there is provided an anti-FAP construct (e.g., a HCAb) variant comprising a variant Fc region comprising one or more amino acid substitutions which increase half-life and/or improve binding to the neonatal Fc receptor (FcRn). Antibodies with increased half-lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol.M-lV) (1994)), are described in US2005/0014934A1 (Hinton et al.). Those antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of Fc region re sidues: 238, 256, 265, 272, 286, 303, 305, 307, 311 , 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).

See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. Nos. 5,648,260; 5,624,821; and WO 94/29351 concerning other examples of Fc region variants.

Anti-FAP constructs (such as HCAb or anti-FAP sd Ab fused to a full-length antibody) comprising any of the Fc variants described herein, or combinations thereof, are contemplated. d) Cysteine Engineered Antibody Variants

In some embodiments, it may be desirable to create cysteine engineered anti-FAP constructs, e.g., “thioMAbs,” in which one or more residues of an antibody are substituted with cysteine residues. In particular embodiments, the substituted residues occur at accessible sites of the antibody. By substituting those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create an im munocon jugate, as described further herein. In some embodiments, any one or more of the following residues maybe substituted with cysteine: Al 18 (EU numbering) of the heavy chain; and 5400 (EU numbering) of the heavy chain Fc region. Cysteine engineered anti-FAP constructs may be generated as described, e.g., in U.S. Pat. No. 7,521,541. e) Antibody Derivatives

In some embodiments, an anti-FAP construct provided herein may be further modified to contain additional nonproteinaceous moieties that are known in the art and readily available. The moieties suitable for derivatization of the antibody include but are not limited to water soluble polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene gl ycol/propyl ene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n- vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer are attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.

In some embodiments, conjugates of an anti-FAP construct and nonproteinaceous moiety that may be selectively heated by exposure to radiation are provided. In some embodiments, the nonproteinaceous moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. Z/54102: 11600-11605 (2005)). The radiation may be of any wavelength, and includes, but is not limited to, wavelengths that do not harm ordinary cells, but which heat the nonproteinaceous moiety to a temperature at which cells proximal to the antibody-nonproteinaceous moiety are killed.

In some embodiments, an anti-FAP construct provided herein (such as anti-FAP sdAb, anti-FAP HCAb, anti- FAP/antiCTLA-4 HCAb, anti-FAP/TIGIT bispecific antibody, anti-FAP/TIM-3 bispecific antibody or anti-FAP/LAG-3 bispecific antibody) may be further modified to contain one or more biologically active protein, polypeptides or fragments thereof. “Bioactive” or “biologically active” as used herein means showing biological activity in the body to carry out a specific function. For example, it may mean the combination with a particular biomolecule such as protein, DNA, etc., and then promotion or inhibition of the activity of such biomolecule. In some embodiments, the bioactive protein or fragments thereof have im munostimula tory/im munoregulatory, membrane transport, or enzymatic activities.

In some embodiments, the bioactive protein or fragments thereof that can be fused with the anti-FAP construct described herein is a ligand, such as lymphokines and cellular factors which interact with specific cellular receptor. Lymphokines are low molecular weight proteins which are secreted by T cells when antigens or lectins stimulate T cell growth. Examples of lymphokines include, but are not limited to, interferon-a, interferon-y, interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-3 (IL-3), tumor necrosis factor (TNF), a colony stimulating factor (e.g. CSF-1, G-CSF or GM-CSF), chemotaxins, macrophage migration inhibitory factor (Ml F), macrophage-activating factor (MAF), NK cell activating factor, T cell replacing factor, leukocyte-inhibitory factor (LIF), lymphotoxins, osteoclast-activating factor (OAF), soluble immune response suppressor (SIRS), growth-stimulating factor, monocyte growth factor, etc. Cellular factors which may be incorporated into the anti-FAP fusion proteins of the invention include but are not limited to tumor necrosis factor a (TN Fa), interferons (IFNs), and nerve growth factor (NGF), etc.

Labels

The anti-FAP constructs of the invention can be labeled. The labels can assist in the identification or detection of the construct. Labels for this purpose are well-known in the art and include elements such as fluorophores, enzymes, and isotopic labels, such as radiolabels [e.g., radionuclides). Various labels, such as radiolabels, methods of labeling, and methods of using radiolabeled agents for therapy and screening are described in US Patent 11,298,433, which is incorporated herein by reference in its entirety.

Pharmaceutical Compositions Further provided by the present application are pharmaceutical compositions comprising any one of the anti-FAP constructs comprising a sdAb specifically recognizing FAP as described herein (such as anti-FAP sd Ab, anti-FAP HCAb, anti- FAP/antiCTLA-4 HCAb, anti-FAP/TIGIT bispecific antibody, anti-FAP/TIM-3 bispecific antibody or anti-FAP/LAG-3 bispecific antibody), and optionally a pharmaceutically acceptable carrier. Pharmaceutical compositions can be prepared by mixing an anti-FAP construct described herein having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions.

The pharmaceutical composition is preferably to be stable, in which the anti-FAP construct comprising anti-FAP sd Ab described here essentially retains its physical and chemical stability and integrity upon storage. Various analytical techniques for measuring protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery, 247-301 , Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10:29-90 (1993). Stability can be measured at a selected temperature for a selected time period. For rapid screening, the formulation may be kept at 40° C. for 2 weeks to 1 month, at which time stability is measured. Where the formulation is to be stored at 2-8° C., generally the formulation should be stable at 30° C. or 40° C. for at least 1 month, and/or stable at 2-8° C. for at least 2 years. Where the formulation is to be stored at 30° C., generally the formulation should be stable for at least 2 years at 30° C., and/or stable at 40° C. for at least 6 months. For example, the extent of aggregation during storage can be used as an indicator of protein stability. In some embodiments, the stable formulation of anti-FAP construct described herein may comprise less than about 10% (preferably less than about 5%) of the anti-FAP construct present as an aggregate in the formulation.

Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers, antioxidants including ascorbic acid, methionine, Vitamin E, sodium metabisulfite; preservatives, isotonicifiers (e.g. sodium chloride), stabilizers, metal complexes (e.g. Zn-protein complexes); chelating agents such as EDFA and/or non-ionic surfactants.

Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDFA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™ or polyethylene glycol (PEG).

Buffers are used to control the pH in a range which optimizes the therapeutic effectiveness, especially if stability is pH dependent. Buffers are preferably present at concentrations ranging from about 50 mM to about 250 mM. Suitable buffering agents for use in the present application include both organic and inorganic acids and salts thereof. For example, citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate. Additionally, buffers may comprise histidine and trimethylamine salts such as Tris.

Preservatives are added to retard microbial growth, and are typically present in a range from 0.2°/o-l .0% (w/v). The addition of a preservative may, for example, facilitate the production of a multi-use (multiple-dose) formulation. Suitable preservatives for use in the present application include octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium halides (e.g., chloride, bromide, iodide), benzethonium chloride; thimerosal, phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben,- catechol; resorcinol; cyclohexanol, 3-pentanol, and m-cresol.

Tonicity agents, sometimes known as “stabilizers” are present to adjust or maintain the tonicity of liquid in a composition. When used with large, charged biomolecules such as proteins and antibodies, they are often termed “stabilizers” because they can interact with the charged groups of the amino acid side chains, thereby lessening the potential for inter and intra-molecular interactions. Tonicity agents can be present in any amount between 0.1% to 25% by weight, preferably 1% to 5%, taking into account the relative amounts of the other ingredients. Preferred tonicity agents include polyhydric sugar alcohols, preferably trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol.

Additional excipients include agents which can serve as one or more of the following: (1 ) bul kin g agents, (2) solubility enhancers, (3) stabilizers and (4) and agents preventing denaturation or adherence to the container wall. Such excipients include: polyhydric sugar alcohols (enumerated above); amino acids such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalan ine, glutamic acid, threonine, etc.,- organic sugars or sugar alcohols such as sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbose, xylose, ribose, ribitol, myoinisitose, myoinisitol, galactose, galactitol, glycerol, cyclitols (e.g., inositol), polyethylene glycol; sulfur containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, a-monothioglycerol and sodium thio sulfate; low molecular weight proteins such as human serum albumin, bovine serum albumin, gelatin or other immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides (e.g., xylose, mannose, fructose, glucose; disaccharides (e.g., lactose, maltose, sucrose); trisaccharides such as raffinose; and polysaccharides such as dextrin or dextran.

Non-ionic surfactants or detergents (also known as “wetting agents”) are present to help solubilize the therapeutic agent as well as to protect the therapeutic protein against agitation-induced aggregation, which also permits the formulation to be exposed to shear surface stress without causing denaturation of the active therapeutic protein or antibody. Non-ionic surfactants are present in a range of about 0.05 mg/ml to about 1.0 mg/ml, preferably about 0.07 mg/ml to about 0.2 mg/ml.

Suitable non-ionic surfactants include polysorbates (20, 40, 60, 65, 80, etc.), polyoxamers (184, 188, etc.), PLURONIC® polyols, TRITON®, polyoxyethylene sorbitan monoethers (TWEEN®-20, TWEEN®-80, etc.), lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, sucrose fatty acid ester, methyl celluose and carboxymethyl cellulose. Anionic detergents that can be used include sodium lauryl sulfate, dioctyle sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

In order for the pharmaceutical compositions to be used for in vivo administration, they must be sterile. The pharmaceutical composition may be rendered sterile by filtration through sterile filtration membranes. The pharmaceutical compositions herein generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

The route of administration is in accordance with known and accepted methods, such as by single or multiple bolus or infusion over a long period of time in a suitable manner, e.g., injection or infusion by subcutaneous, intravenous, intraperitoneal, intramuscular, intra-arterial, intralesional or intraarticular routes, topical administration, inhalation or by sustained release or extended-release means. In some embodiments, the pharmaceutical composition is administered locally, such as intratumorally.

Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing the antagonist, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hyd roxy ethy l-m ethacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No.3,773,919), copolymers of L-glutamic acid and. ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRO N DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly- D-(— )-3-hydroxybu tyric acid.

The pharmaceutical compositions herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition may comprise a cytotoxic agent, chemotherapeutic agent, cytokine, immunosuppressive agent, or growth inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.

The active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or g elatin -m icrocap sule s and poly-(me thyl methacy late) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 18t edition.

Methods of Treating FAP-Related Diseases

The anti-FAP construct comprising sdAb specifically recognizing FAP as described herein (such as anti-FAP sdAb, anti- FAP H C Ab, anti-FAP/antiCTLA-4 HCAb, anti-FAP/TIGIT bispecific antibody, anti-FAP/TI A/l-3 bispecific antibody or anti- FAP/LAG-3 bispecific antibody), and the compositions (such as pharmaceutical compositions) thereof are useful for a variety of applications, such as in diagnosis, molecular assays, and therapy.

One aspect of the invention provides a method of treating a FAP-related disease or a condition in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of a pharmaceutical composition comprising the anti-FAP construct described herein.

FAP-related diseases include diseases associated with FAP expression (e.g., aberrant FAP expression). Exemplary FAP-related diseases include cancer, fibrosis, arthritis, atherosclerosis, autoimmune diseases, metabolic diseases. See, e.g., Fitzgerald et al. 2020 (Fitzgerald AA, Weiner LM. The role of fibroblast activation protein in health and malignancy. Cancer Metastasis Rev.2020 Sep;39(3):783-803)

The present invention contemplates, in part, protein constructs (such as anti-FAP sdAb, anti-FAP HCAb, anti- FAP/antiCTLA-4 HCAb, anti-FAP/TIGIT bispecific antibody, anti-FAP/TIM-3 bispecific antibody or anti-FAP/LAG-3 bispecific antibody), nucleic acid molecules and/or vectors encoding thereof, host cells comprising nucleic acid molecules and/or vectors encoding thereof, that can be administered either alone or in any combination with another therapy, and in at least some aspects, together with a pharmaceutically acceptable carrier or excipient. In some embodiments, prior to administration of the anti-FAP construct, they may be combined with suitable pharmaceutical carriers and excipients that are well known in the art. The compositions prepared according to the disclosure can be used for the treatment or delaying of worsening of cancer.

In some embodiments, there is provided a method of treating cancer comprising administering to the individual a therapeutically effective amount of a pharmaceutical composition comprising an isolated anti-FAP construct comprising a single-domain antibody (sdAb) moiety specifically recognizing FAP (such as anti-FAP sd Ab, anti-FAP H C Ab, anti-FAP/an tiCTLA-4 HCAb, anti-FAP/TI GIT bispecific antibody, anti-FAP/TIM-3 bispecific antibody or anti-FAP/LAG-3 bispecific antibody). In some embodiments, the cancer is a solid tumor (such as colon cancer). In some embodiments, the pharmaceutical composition is administered systemically (such as intravenously). In some embodiments, the pharmaceutical composition is administered locally (such as intratumorally). In some embodiments, the method further comprises administering to the individual an additional cancer therapy (such as surgery, radiation, chemotherapy, immunotherapy, hormone therapy, or a combination thereof). In some embodiments, the individual is a human. In some embodiments, the method of treating cancer has one or more of the following biological activities: (1) killing cancer cells (including bystander killing); (2) inhibiting proliferation of cancer cells; (3) inducing immune response in a tumor; (4) reducing tumor size; (5) alleviating one or more symptoms in an individual having cancer,- (6) inhibiting tumor metastasis,- (7) prolonging survival; (8) prolonging time to cancer progression,- and (9) preventing, inhibiting, or reducing the likelihood of the recurrence of a cancer. In some embodiments, the method of killing cancer cells mediated by the pharmaceutical composition described herein can achieve a tumor cell death rate of at least about any of 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. In some embodiments, the method of killing cancer cells mediated by the pharmaceutical composition described herein can achieve a bystander tumor cell (uninfected by the oncolytic VV) death rate of at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. In some embodiments, the method of reducing tumor size mediated by the pharmaceutical composition described herein can reduce at least about 10% (including for example at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%) of the tumor size. In some embodiments, the method of inhibiting tumor metastasis mediated by the pharmaceutical composition described herein can inhibit at least about 10% (including for example at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%) of the metastasis. In some embodiments, the method of prolonging survival of an individual (such as a human) mediated by the pharmaceutical composition described herein can prolongs the survival of the individual by at least any of 1,2, 3,4,5, 6, 7, 8, 9, 10, 11, 12, 18, or 24 months. In some embodiments, the method of prolonging time to cancer progression mediated by the pharmaceutical composition described herein can prolongs the time to cancer progression by at least any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks.

The methods described herein are suitable for treating a variety of cancers, including both solid cancer and liquid cancer. The methods are applicable to cancers of all stages, including early stage cancer, non-metastatic cancer, primary cancer, advanced cancer, locally advanced cancer, metastatic cancer, or cancer in remission. The methods described herein may be used as a first therapy, second therapy, third therapy, or combination therapy with other types of cancer therapies known in the art, such as chemotherapy, surgery, hormone therapy, radiation, gene therapy, immunotherapy (such as T-cell therapy), bone marrow transplantation, stem cell transplantation, targeted therapy, cryotherapy, ultrasound therapy, photodynamic therapy, radio-frequency ablation or the like, in an adjuvant setting or a neoadjuvant setting (i.e., the method may be carried out before the primary/definitive therapy). In some embodiments, the method is used to treat an individual who has previously been treated. In some embodiments, the cancer has been refractory to prior therapy. In some embodiments, the method is used to treat an individual who has not previously been treated. In some embodiments, the method is suitable for treating cancers with aberrant FAP expression, activity and/or signaling include, by way of non-limiting example, melanoma, prostate cancer, lung cancer, colon cancer, gastric cancer, ovarian cancer, breast cancer, and glioblastoma.

In some embodiments, there is provided a method of treating an immunotherapy-responsive solid tumor (such as carcinoma or adenocarcinoma, such as cancers with aberrant FAP expression, activity and/or signaling), comprising administering to the individual a therapeutically effective amount of a pharmaceutical composition comprising an isolated anti- FAP construct comprising a sd Ab moiety specifically recognizing FAP (such as anti-FAP sd Ab, anti-FAP H C Ab, a nti-F AP/an tiCTLA- 4 HC Ab, an ti- FAP/TI GIT bispecific antibody, anti-F AP/TI M-3 bispecific antibody or anti-FAP/LAG-3 bispecific antibody).

Some cancers whose growth may be inhibited using the antibodies of the invention include cancers typically responsive to immunotherapy. Non-limiting examples of other cancers for treatment include melanoma (e.g., metastatic malignant melanoma), renal cancer (e.g. clear cell carcinoma), prostate cancer (e.g. hormone refractory prostate adenocarcinoma), breast cancer, colon cancer and lung cancer (e.g. non-small cell lung cancer). Additionally, the invention includes refractory or recurrent malignancies whose growth may be inhibited using the antibodies of the invention. Examples of other cancers that may be treated using the antibodies of the invention include bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, chronic or acute leukemias including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, and combinations of said cancers. The present invention is also useful for treatment of metastatic cancers, especially metastatic cancers that express FAP. In some embodiments, the methods described herein are suitable for treating a colorectal cancer, such as adenocarcinoma, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, Leiomyosarcoma, melanoma, or squamous cell carcinoma.

Dosages and desired drug concentrations of pharmaceutical compositions of the present application may vary depending on the particular use envisioned. The determination of the appropriate dosage or route of administration is well within the skill of an ordinary artisan. Animal experiments provide reliable guidance for the determination of effective doses for human therapy. Interspecies scaling of effective doses can be performed following the principles laid down by Mordenti, J. and Chappell, W. “The Use of Interspecies Scaling in Toxicokinetics,” In loxicokinetics and New Drug Development, Yacobiet al., Eds, Pergamon Press, New York 1989, pp.42-46.

When in vivo administration of the anti-FAP construct comprising an anti-FAP sdAb moiety described herein are used, normal dosage amounts may vary from about 10 ng/kg up to about 100 mg/kg of mammal body weight or more per day, preferably about 1 mg/kg/day to 10 mg/kg/day, such as about 1 -3 mg/kg/day, about 2-4 mg/kg/day, about 3-5 mg/kg/day, about 4-6 mg/kg/day, about 5-7 mg/kg/day, about 6-8 mg/kg/day, about 6-6.5 mg/kg/day, about 6.5-7 mg/kg/day, about 7-9 mg/kg/day, or about 8-10 mg/kg/day, depending upon the route of administration. It is within the scope of the present application that different formulations will be effective for different treatments and different disorders, and that administration intended to treat a specific organ or tissue may necessitate delivery in a manner different from that to another organ or tissue. Moreover, dosages may be administered by one or more separate administrations, or by continuous infusion. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

In some embodiments, the pharmaceutical composition is administered for a single time (e.g. bolus injection). In some embodiments, the pharmaceutical composition is administered for multiple times (such as any of 2, 3, 4, 5, 6, or more times). If multiple administrations, they may be performed by the same or different routes and may take place at the same site or at alternative sites. The pharmaceutical composition may be administered twice per week, 3 times per week, 4 times per week, 5 times per week, daily, daily without break, once per week, weekly without break, once per 2 weeks, once per 3 weeks, once per month, once per 2 months, once per 3 months, once per 4 months, once per 5 months, once per 6 months, once per 7 months, once per 8 months, once per 9 months, once per 10 months, once per 11 months, or once per year. The interval between administrations can be about any one of 24 h to 48 h, 2 days to 3 days, 3 days to 5 days, 5 days to 1 week, 1 week to 2 weeks, 2 weeks to 1 month, 1 month to 2 months, 2 month to 3 months, 3 months to 6 months, or 6 months to a year. Intervals can also be irregular (e.g. following tumor progression). In some embodiments, there is no break in the dosing schedule. In some embodiments, the pharmaceutical composition is administered every 4 days for 4 times. The optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.

The pharmaceutical compositions of the present application, including but not limited to reconstituted and liquid formulations, are administered to an individual in need of treatment with the anti-FAP construct described herein, preferably a human, in accord with known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intravenous (i.v.), intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. A reconstituted formulation can be prepared by dissolving a lyophilized anti- FAP construct described herein in a diluent such that the protein is dispersed throughout. Exemplary pharmaceutically acceptable (safe and non-toxic for administration to a human) diluents suitable for use in the present application include, but are not limited to, sterile water, bacteriostatic water for injection (BWFI), a pH buffered solution (e.g. phosphate-buffered saline), sterile saline solution, Ringer's solution or dextrose solution, or aqueous solutions of salts and/or buffers.

In some embodiments, the pharmaceutical compositions are administered to the individual by subcutaneous (i.e. beneath the skin) administration. For such purposes, the pharmaceutical compositions may be injected using a syringe. However, other devices for administration of the pharmaceutical compositions are available such as injection devices; injector pens,- auto-injector devices, needleless devices,- and subcutaneous patch delivery systems.

In some embodiments, the pharmaceutical compositions are administered to the individual intravenously. In some embodiments, the pharmaceutical composition is administered to an individual by infusion, such as intravenous infusion. Infusion techniques for immunotherapy are known in the art (see, e.g., Rosenberg et al., New Eng. J. of Med.319: 1676 (1988)).

Methods of Preparation The anti-FAP construct (such as anti-FAP single-domain antibodies) described herein may be prepared using any methods known in the art or as described herein. Also see the following examples.

Methods of preparing single-domain antibodies have been described. See, for example, Els Pardon et al, Nature Protocol, 2014; 9(3): 674. Single-domain antibodies (such as VHHs) may be obtained using methods known in the art such as by immunizing a Camelid species (such as camel or llama) and obtaining hybridomas therefrom, or by cloning a library of singledomain antibodies using molecular biology techniques known in the art and subsequent selection by ELISA with individual clones of unselected libraries or by using phage display.

For recombinant production of the single-domain antibodies, the nucleic acids encoding the single-domain antibodies are isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for expression. D N A encoding the single-domain antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody). Many vectors are available. The choice of vector depends in part on the host cell to be used. Generally, preferred host cells are of either prokaryotic or eukaryotic (generally mammalian) origin.

The elements and method steps described herein can be used in any combination whether explicitly described or not.

All combinations of method steps as used herein can be performed in any order, unless otherwise specified or clearly implied to the contrary by the context in which the referenced combination is made.

As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise.

Numerical ranges as used herein are intended to include every number and subset of numbers contained within that range, whether specifically disclosed or not. Further, these numerical ranges should be construed as providing support for a claim directed to any number or subset of numbers in that range. For example, a disclosure of from 1 to 10 should be construed as supporting a range of from 2 to 8, from 3 to 7, from 5 to 6, from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, and so forth.

All patents, patent publications, and peer-reviewed publications (i.e., “references”) cited herein are expressly incorporated by reference to the same extent as if each individual reference were specifically and individually indicated as being incorporated by reference. In case of conflict between the present disclosure and the incorporated references, the present disclosure controls.

It is understood that the invention is not confined to the particular construction and arrangement of parts herein illustrated and described, but embraces such modified forms thereof as come within the scope of the claims.

EXAMPLES

Introduction

Camelid antibodies also known as nanobodies of variable-heavy-heavy domains (VHHs) were used to develop a targeted agent for fibroblast activation protein (FAP). Traditional immunoglobulins (IgGs) exhibit poor pharmacokinetics - slow tumor uptake, poor tissue penetration, and delayed clearance from the blood. This results in the prolonged exposure of nontarget tissue to radioactivity and adversely affects the therapeutic index of radioimmunotherapy. A low molecular weight antibody scaffold, such as a VHH with faster tumor uptake, greater tissue penetration, and rapid clearance from the blood would be predicted to result in decreased irradiation of non-target tissue leading to an increase in therapeutic efficacy. VHH binding domains are single-domain fragments composed entirely of heavy chains (Bathula et al.2021). VHHs have three CDRs for target engagement as opposed to the six CDRs (3 heavy, 3 light) found in human antibodies. However, the CDR3 of camelid VHHs can be over 20 amino acid residues long (Steeland et al. 2016). This provides them with a unique architecture and molecular dexterity that allows them to bind epitopes inaccessible to conventional antibodies (cryptic epitopes) with unparalleled affinity. VHHs are easy to produce in large quantities because of their high solubility and require little to no humanization due to their high homology with human heavy chains. Here, we present the discovery of a novel VHH for FAP (termed F7 VHH) and its use as a PET imaging agent as a monomer, tethered dimer (F7-D), and Fc fusion protein (F7-Fc).

Methods

F7 Lead Identification

In order to identify a high-affinity FAP-targeting camelid VHH, a phage biopanning campaign was employed. Using an initial lead camelid VHH (Fl VHH), an affinity maturation process was employed using a controlled mutational scheme via GenART (ThemoFisher). A library of variants based on Fl VHH was made with a size of 10^B clones with an average amino acid mutational rate of 1 in the CDR1 region and 4 in the CDR3 region. The library of variants was packaged into a phagemid (P ADL22 c, Antibody Design Labs) and using Ml 3K07 Helper Phage the display library was rescued from F. coiiwA the resulting VHH phage display library was used for biopanning. Biopanning followed previously established protocols (Ye et al.2020) and was carried out for two rounds with clones screened following each round.

To screen potential clones for binding capabilities, an ELISA based method was employed. FAP-targeted VHH clones were produced from a total of 864 monoclonal colonies using 5 mM IPTG induction in a 96- well growth plate. Using a SS320 F. co// strain, the VHH clones were present in the supernatant following induction and overnight incubation at 30 °C with 250 rpm. Supernatant of each VHH clone was then used in a FAP ELISA. The ELISA followed previously described methods. In brief, MaxiSorp Nunc plates were coated with 5 |Jg/mL of streptavidin overnight at 4 °C. The wells were washed twice with PBS before the wells were blocked with 370 |Jl of 2% BSA for 1 h at room temperature on a small radius shaker with 300 rpm. Unless noted, all additional incubations were at room temperature with 300 rpm shaking. A subsequent wash was performed three times with PBST (0.005% Tween20). Recombinant FAP (ACRO) was added to the wells at 1 |Jg/mL in PBST with 1% BSA and allowed to incubate for 1 h. Wells were washed 3 times again to remove any unbound FAP before the supernatants of the VHH clones was added to the wells diluted in PBST with 1% BSA and allowed to incubate for Ih. Wells were washed again with PBST 3 times before an anti-HA secondary antibody with HRP (Sigma Aldrich 12-013-819-01) conjugated to it was added in a 1:1000 dilution in PBST with 1% BSA was added to each well and incubated for 1 h. For the final visualization of VHH binding, the wells were washed three times with PBST before 50 pl of Turbo TMB reagent (Pierce) was added to each well. The peroxidase reaction was allowed to occur for no longer than 5 minutes and was stopped with 10 |Jl of I M H₂SO₄. The absorbance at 450 nm was read using a microplate reader. Positive clones underwent a second ELISA using a dilution series of induced supernatant and the top 24 hits were sequenced to identify unique clones.

Protein Purification

The VHH phagemid was cloned into a modified pet22b expression vector to include a TEV protease cleavable site to remove the 6x His purification tag. The plasmid containing was cloned into Shuffle T7 f. ro/zcells and grown in TB media. For purification IL of F7 VHH-containing F. coii'UK grown to DD600 of 0.8 at 37 °C with 250 rpm before being inoculated with 10 mM I PTG and once induced was grown at 16 °C with 250 rpm overnight. The following day, the F7 VHH was purified using a periplasmic prep method. Briefly the cells were spun down at 7500 x g for 15 min and supernatant was removed. The cell pellet was resuspended in 30 ml of TES buffer and slowly stirred for 30 minutes at 4 °C. The cells were spun again at 7500 x g for 30 minutes and the pellet was resuspended in 30 ml of Tris Buffer and stirred slowly at 4 °C for 30 minutes. The cells went through a final spin at 7500 x g for 30 minutes. The supernatant containing F7 VHH was collected and captured via a 5- mL HiTrap HP His column (cytivia) before an imidazole purification was run on an AKTA HPLC. The purified F7 VHH was collected and TEV protease was used to cleave the affinity tags and the sample was rerun over the His column, with flowthrough collected, and the final protein sample was dialyzed into PBS.

A F7 dimer (F7-D) was produced in a similar fashion with the change that the modified plasmid contained a 5x GS4 linker ((GGGGS)₅ (SEQ ID N0:87)) with the F7 VHH cloned onto either flanking side of the linker and a N-terminus TEV cleavable SUMO tag with a 6x His tag. For the F7-D once purified via a 5-mL HiTrap HP His column, the protein was combined with a TEV protease to remove the 6xHis-SUMO tag and construct was purified again collecting the flowthrough of the 5-mL HiTrap HP His column followed by size exclusion chromatography (HiLoad Superdex 75 10/300 (Cytiva)).

Vectors for mammalian expression of an F7 VHH-Fc fusion (F7-Fc) were generated by Genscript. Briefly, codon optimized sequences encoding F7 VHH were synthesized de novo and subcloned into TGEX-SCblue (Antibody Design Labs, San Diego, CA) in frame with the PelBK leader sequence (ETDTI LLWVLLLLAAQPAMA; SEQ ID N 0 :88) (Valadon et al.2006) and a human IgGl Fc sequence (SEQ ID N 0 :81 ), using standard molecular biology methods. Constructs were transformed into competent NEB 5al pha cells (New England Biolabs) and plasmid DNA was purified using Pure Link HiPure plasmid maxiprep kits (Thermo Fisher) according the vendor's recommended protocol. For F7-Fc protein production, ExpiCHO-S cells were maintained in ExpiCHO Expression Medium at 37°C and 8% CO₂, shaking at 120 rpm on a 25-mm orbital diameter in vented Erlenmeyer flasks. Cells were cultured to a density of 6 x 10⁶ cell s/m I with a viability >95%, and were transfected with F7-TGEX-SCblue. Transfections were performed using an Expifectamine CHO Transfection Kit [Thermo Fisher) according to the vendor's recommended protocol, using 1 |Jg of plasmid DNA and 3.2 |J I of Expifectamine CHO Reagent per ml of suspension culture. Cells were returned to 37 °C and 8% C0₂ incubator overnight, shaking at 120 rpm. The following day, transfected cells were supplemented with 6 |Jl of ExpiCHO Enhancer and 240 pl of ExpiCHO Feed per ml of suspension culture, and incubator conditions were changed to 32 °C and 5% CO₂. 12 days post-transfection, suspension cultures were harvested and centrifuged at 2000 x RCF for 10 min at 4 °C. The protein-containing supernatant was collected and further clarified by centrifuging at 20,000 x RCF for 30 min at 4 °C and passed through a 0.22-JJ m sterile filter. To purify the F7-Fc antibody, it was captured using protein A and were further purified by size exclusion chromatography. A HiTrap Protein A HP column (Cytiva) was equilibrated with 5 column volumes (CVs) of PBS. Clarified ExpiCHO-S supernatant was supplemented with 300 mM NaCI, pH was adjusted to 6.8, and was pumped onto the protein A column at 1 CV/min. Unbound protein was washed from the column using 10 CVs of PBS. F7-Fc was eluted in 2.5 CVs of 100 mM glycine pH 3.0, followed by 2.5 CVs of PBS. Eluate was immediately neutralized using 2M Tris HCI pH 8.6. Eluate was concentrated and buffer exchanged into PBS using an Amicon stirred chamber with a 30-kDa MWCO ultrafiltration membrane (Millipore). Size exclusion chromatography was performed on an AKTApure fast protein liquid chromatography system using a HiLoad 16/600 Superdex 200 PG (Cytiva) column equilibrated with PBS. Samples were loaded and fractionated using a mobile phase of PBS at 0.5 ml/min, chromatograms were obtained by monitoring UV absorbance at 280 nm. Eluted protein fractions contributing to a single peak of UV absorbance corresponding to the theoretical molecular mass of F7-Fc were collected and pooled. Eluates were diluted to I mg/ml in PBS, dispensed into 0.5 -ml aliquots, and flash frozen. Bio-Layer Interferometry (BL1)

Bio-layer interferometry (BLI) measurements were obtained using a Sartorius Octet R8. BLI experiments followed established protocols (Ye et al. 2020, Hintz et al. 2019). Briefly, biotinylated human FAP protein (ACRO) was captured on hydrated SAX (high precision streptavidin) biosensors. A assay buffer of PBS with 1% BSA was used for all steps including dilutions. Hydrated SAX biosensors were equilibrated for 30 s in the assay buffer before the biotinylated FAP was captured for 45 s. A second equilibration step was carried out for 30 s. Serial dilutions of the various anti-FAP constructs were then exposed to the FAP loaded SAX biosensor for 180-240 s followed by a dissociation step in assay buffer for an equivalent amount of time. Two separate controls were run to evaluate non-specific binding. One control included a FAP loaded SAX biosensor exposed to assay buffer in both association and dissociation steps. A second control included a SAX biosensor with no FAP loaded exposed to the highest concentration of the anti-FAP constructs. The no-antibody control was subtracted from the data before modeling and the dissociation constant KD was calculated. Graphs were drawn using the corrected data in GraphPad Prism.

Animal Studies

Animal studies were performed under guidelines approved by the University of Wisconsin RARC . All mice in the study were athymic nude-Focnl nu (Envigo). For subcutaneous xenografts, 1 E6 cells were injected in a 1 :1 PBS and Ma trigel (Corning) mixture and injected into either the area above the hind leg or above the animal's shoulder. Animals were split evenly between the FAP positive group receiving CWR-Rl-EnzR-FAP cells or the negative group receiving the parental CWR-R1 -EnzR cells. Tumors were allowed to grow to at least 300 mm³ before initiation of the imaging studies.

Nuclear Imaging Study

For the nuclear imaging studies in this project either⁶⁴Cu or⁶⁸Ga was used and chelation addition and radioconjugations followed established protocols. The⁶⁴Cu was provided by the University of Wisconsin Medical Physics Department (Madison, Wl) and the University of Wisconsin-Madison Cyclotron Research Group. Following p-SC N-Bn-N 0 TA (NOTA, Macrocyd ics) addition to the anti-FAP constructs (F7 VHH, F7-D, F7-Fc),⁶⁴Cu was conjugated in a NaOAC buffered reaction. The⁶⁴Cu was resuspended in pH 5.0 NaOAc buffer. The anti-FAP constructs were conjugated at the following ratios 25 |Jg F7 VHH, 50 |Jg F7-D, and 100 |Jg F7-Fc for every 3.7 MBq. The anti-FAP constructs were added to the buffered⁶⁴Cu and allowed to incubate at 37 °C with gentle agitation. To finish the conjugation Tween-20 was added to a final concentration of 0.005% and the conjugated constructs were purified using a size exclusion PD-10 column preequilibrated with PBS buffer.

All microPET/CT studies were performed on an Inveon |JPET/CT Scanner (Siemens Medical Solutions). For the comparison of F7 VHH and FAPI -46, small molecule mice (n=3 for each experimental and control groups) were injected with⁶⁸Ga-labeled FAPI-46 (5.986 MBq Average) via tail vein injection. One hour post injection, mice were anesthetized via inhalation of 2.5% isoflurane and PET/CT images were acquired. The same mice were then injected the next day with⁶⁴Cu- labele d F7 VHH (5.688 MBq average) and a 1 h dynamic scan was performed followed by a second image acquisition at 4 h post injection. PET list mode data were acquired for 80 million counts using a gamma ray energy window of 350-650 KeV and a coincidence timing window of 3.438 ns. A CT-based attenuation correction was performed for approximately 10 minutes with 80 kVp, 1 mA, 220 rotation degrees in 120 rotation steps, 250 ms exposure time, and subsequently reconstructed using a Shepp-Logan filter with 210 micron isotropic voxels. Scons were reconstructed using 3-dimensionol ordered-subset expectation maximization (2 iterations, 16 subsets) with a maximum a posteriori probability algorithm (0SEM3DMAP). Two-dimension (2D) images and maximum intensity projections (MIPs) were prepared in Inveon Research Workplace and ROIs were manually drawn and quantified in Inveon Research Workspace.

The |JPET/CT studies for the F7-D dimer and the F7-Fc fusion followed the same protocols as above with the only variation being the acquisition times for the scans. F7-D had images acquired at 1 , 4, and 24 h post injection of⁶⁴Cu labeled F7-D (8.723 MBq average). While F7-Fc had image acquisition performed at 4, 24, and 48h post injection of labeled F7-Fc (9.922MBq average). All image quantification was the same as described above. All graphs were drawn using GraphPad Prism.

Results

To identify an anti-FAP VHH for PET imaging, we utilized antibody phage display rather than direct immunization of a live animal. A naive VHH phage display library - (diversity of 7.5 x IO¹⁰) developed in-house - was screened against recombinant FAP on magnetic beads. After four rounds of biopanning, we identified 29 clones that bound FAP by ELISA. Out these clones, seven unique sequences were identified (NbA-Nb G VHHs). All seven of the clones were specific for FAP by ELISA and did not cross-react with DPPIV. DPPIV is a member of the prolyl protease family and shares 70% amino acid sequence homology with FAP. Based on ELISA data, a lead VHH, NbC VHH, was selected. The K_D of NbC VHH was determined to be 78 nM by biolayer interferometry (BLI) (Table 7). Affinity maturation was then performed on NbC VHH by site-specific mutagenesis at four amino acids in CDR3 and one amino acid in CDR1 . Two additional rounds of biopanning against FAP were performed using the affinity matured sub-library with a diversity of 1.4 x 10⁹. A screen of 384 clones identified 93 (24.7%) as strong binders through a stringent ELISA. Out of the 27 highest binding clones, 24 were revealed to be unique. Clone F7 was selected as our lead for further investigation based on its affinity for FAP (K_D - 7 nM, Table 7) and ease of expression in bacteria (>30 mg/L yield). F7 was engineered into homodimer (F7-D) using a (G₄S)₅ linker and grafted onto a human Fc domain to create a VHH-Fc fusion protein (F7-Fc). The K_D values for the bivalent constructs were found to be 29 pM for F7-D and 38 pM for F7-Fc, respectively, as measured by BLI (Table 7). Sequences for various of the above-referenced constructs are provided in Tables 3-6.

Table 3. Complementary determining regions (CDRs) of exemplary sdAbs.

ID: SEQ ID NO Table 4. Framework Regions 1 (FR1) and 2 (FR2) of exemplary sdAbs.

D: SEQ ID NO Table 5. Framework Regions 3 (FR3) and 4 (FR4) of exemplary sdAbs.

D: SEQ ID NO

Table 6. Exemplary sdAbs.

D: SEQ ID NO

Table 7. Affinity constants of anti-FAP constructs as determined by BLI.

)ata is shown as M with SD.

F7-Fc showed specific binding to a prostate cancer cell line engineered to over express FAP (CWR-R1^FAP) compared to the parental cell line by radioligand binding assay and flow cytometry (FIGS.2A-2D).

We next compared the ability of our monovalent F7VHH to F API -46 at detecting FAP expression in a xenograft model. Mice bearing FAP-expressing CWR-R1FAP or FAP null CWR-R1 xenografts were administered either⁶⁸Ga-labeled FAPI-46 ([⁶⁸Ga]Ga-FAPI-46)or⁶⁴Cu-labeled F7VHH ([⁶⁴Cu]Cu-F7 VHH)(FIGS.3A and 3B). As anticipated fora small-molecule and a protein with a molecular weight of 13 kDa, the uptake for both imaging agents was rapid, with image acquisition possible for both at the 1 h post injection timepoint. [⁶⁸Ga]Ga-FAPI-46 demonstrated rapid uptake in the FAP-expressing xenograft with a %l D/g of 0.58% +/- 0.143 at 1 h. Our biologic [⁶⁴Cu]Cu-F7 VHH had tumor uptake two-fold higher than FAPI-46 in the same xenograft model with an average uptake of 1.1% +/- 0.173 in at 1 h post injection. [⁶⁴Cu]Cu-F7 VHH was also investigated at 4h and have an average uptake of 0.923% +/- 0.125. The MIPs from these scans were consistent with both ROI and ex vivo analysis. The blood clearance of [⁶⁸Ga]Ga-FAPI-46 was rapid with an in vivo ROI of only 0.104% +/- 0.016. Similarly, [⁶⁴Cu]Cu-F7 VHH had a blood uptake ROI of 0.357 +/- 0.085 at 1 h and 0.196% +/-0.103 at 4 h. The [⁶⁴Cu]Cu-F7 VHH had expectedly high signal in the kidneys, 39.367% at 1 h and 29.567% at 4 h.

The higher molecular weight F7 VHH constructs, F7-D Dimer and F7-Fc were next investigated for their ability to image FAP in xenografts by PET. For this study, we decided to use copper-64 as our imaging isotope to be consistent among the different constructs tested. Mice bearing CWR-R1 FAP and C WR-R1 xenografts were injected with [⁶⁴Cu]Cu-F7-D or [⁶⁴Cu] Cu- F7-Fc.

[⁶⁴CU]CU-F7-D was imaged at 1 h, 4 h, and 24 h post-injection given its lower molecular weight of 28 kDa. Tumor uptake was rapid with the [⁶⁴Cu]Cu-F 7-D in FAP-positive tumors having average uptake values of 1 Abl+/- 0.240, 1.433 +/- 0.176, and 1.267 +/- 0.133 at 1, 4, 24 h post-injection respectively (FIGS. 4A-4D). Antithetically, in the FAP null xenografts, tumor uptake was 0.581 +/- 0.079, 0.473+/- 0.041, 0.296 +/- 0.030 again at 1, 4,24 h post-injection. Tumor uptake increased with each later scan as [⁶⁴ Cu] Cu-F7-D was retained in the FAP positive tumors and cleared from the FAP null tumors. Again, the MIP data were consistent with the in i+raand exrivo uptake data. The blood clearance of [⁶⁴Cu]Cu-F7-D was rapid with the blood peaking at 1 h post-injection, 1.867% +/- 0.067, and was four-fold lower than the tumor uptake at 24 h with an average uptake of 0.305% +/- 0.048.

Compared to F7 VHH and F7-D, the largest construct radiolabeled with⁶⁴Cu ([⁶⁴Cu]C u-F 7- Fc) demonstrated the highest uptake in FAP-expressing tumors even at 4 h post-injection (FIGS.5A-5C). At 4 h post-injection, the tumor uptake in FAP positive was 7.5% +/- 1.901. At the 24 and 48 h timepoints, retention and accumulation of [⁶⁴Cu]Cu-F7-Fc was observed with tumor uptake of 15.23% +/- 2.24 and 14.93% +/- 1.24 respectively. Blood clearance was also apparent with values of 5.93% +/- 1.10 and 4.20% +/- 1.32 at 24 and 48 h post-injection. The most significant difference between [⁶⁴Cu]Cu-F7-D and [⁶⁴Cu]Cu-F7- Fc can be seen in the uptake of the liver and kidney. [⁶⁴Cu]Cu-F 7- D had the high uptake in the kidney with 45.53% +/- 4.04 at 4 h post-injection and 12.133% +/- 5.50 at 24 h. Whereas the kidney uptake with [⁶⁴Cu]Cu-F7-Fc peaked at 6.63% +/- 0.338 at 4 h and was 3.40% +/- 0.611 at 24 h. In contrast, the liver uptake for [⁶⁴Cu]Cu-F7-D was 3.06% +/- 0.176 at 4h and 2.30% +/- 0.265 at 24 h. Given its larger size and presence of an Fc domain, the liver uptake of [⁶⁴Cu] Cu-F7-Fc was greater: 11.43% +/- 0.233 at 4 h and 6.40% +/- 0.25 at 24 h.

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Claims

What is claimed is:

1. An isolated anti-FAP construct comprising a single-domain antibody (sd Ab ) moiety specifically recognizing FAP, wherein the sd Ab moiety comprises a CDR1 , a CDR2, and a CDR3, wherein: the CDR1 comprises the amino acid sequence of any one of SEQ ID NOS:1 -8, or a variant thereof comprising up to 3 (such as any of 1, 2, or 3) amino acid substitutions; the C DR2 comprises the amino acid sequence of any one of SEQ ID NOS:9- 16, or a variant thereof comprising up to 3 (such as any of 1, 2, or 3) amino acid substitutions; and/or the CDR3 comprises the amino acid sequence of any one of SEQ ID NOS:17-24, or a variant thereof comprising up to 3 (such as any of 1, 2, or 3) amino acid substitutions.

2. The isolated anti-FAP construct of any prior claim, wherein: the CDR1 comprises the amino acid sequence of any one of SEQ ID NOS:1 -8, or a variant thereof comprising up to 3 (such as any of 1, 2, or 3) amino acid substitutions; the C DR2 comprises the amino acid sequence of any one of SEQ ID NOS:9- 16, or a variant thereof comprising up to 3 (such as any of 1, 2, or 3) amino acid substitutions,- and the CDR3 comprises the amino acid sequence of any one of SEQ ID NOS:17-24, or a variant thereof comprising up to 3 (such as any of 1, 2, or 3) amino acid substitutions.

3. The isolated anti-FAP construct of any prior claim, wherein: the CDR1 comprises the amino acid sequence of any one of SEQ ID NOS:1 -8; the CDR2 comprises the amino acid sequence of any one of SEQ ID NOS:9-16; and/or the CDR3 comprises the amino acid sequence of any one of SEQ ID NOS:17-24.

4. The isolated anti-FAP construct of any prior claim, wherein: the CDR1 comprises the amino acid sequence of any one of SEQ ID NOS:1 -8; the CDR2 comprises the amino acid sequence of any one of SEQ ID NOS:9-16; and the CDR3 comprises the amino acid sequence of any one of SEQ ID NOS:17-24.

5. The isolated anti-FAP construct of any prior claim, wherein: the C DR1 comprises the amino acid sequence of SEQ ID N 0: 1 or a variant thereof comprising up to 3 (such as any of 1 , 2, or 3) amino acid substitutions, the C DR2 comprises the amino acid sequence of SEQ ID N 0 :9 or a variant thereof comprising up to 3

(such as any of 1, 2, or 3) amino acid substitutions, and the CDR3 comprises the amino acid sequence of SEQ ID N0:l 7 or a variant thereof comprising up to 3 (such as any of 1 , 2, or 3) amino acid substitutions,- the C DR1 comprises the amino acid sequence of SEQ ID N 0:2 or a variant thereof comprising up to 3 (such as any of 1 , 2, or 3) amino acid substitutions, the CDR2 comprises the amino acid sequence of SEQ ID N0:l 0 or a variant thereof comprising up to 3 (such as any of 1, 2, or 3) amino acid substitutions, and the CDR3 comprises the amino acid sequence of SEQ ID N0:l 8 or a variant thereof comprising up to 3 (such as any of 1 , 2, or 3) amino acid substitution s; the C D R 1 comprises the amino acid sequence of SEQ ID N 0:3 or a variant thereof comprising up to 3 (such as any of 1 , 2, or 3) amino acid substitutions, the CDR2 comprises the amino acid sequence of SEQ ID N 0:11 or a variant thereof comprising up to 3 (such as any of 1, 2, or 3) amino acid substitutions, and the CDR3 comprises the amino acid sequence of SEQ ID N0:l 9 or a variant thereof comprising up to 3 (such as any of 1 , 2, or 3) amino acid substitutions,- the C DR1 comprises the amino acid sequence of SEQ ID N 0:4 or a variant thereof comprising up to 3 (such as any of 1 , 2, or 3) amino acid substitutions, the CDR2 comprises the amino acid sequence of SEQ ID N 0:12 or a variant thereof comprising up to 3 (such as any of 1, 2, or 3) amino acid substitutions, and the CDR3 comprises the amino acid sequence of SEQ ID N0:2Q or a variant thereof comprising up to 3 (such as any of 1 , 2, or 3) amino acid substitutions,- the C DR1 comprises the amino acid sequence of SEQ ID N 0:5 or a variant thereof comprising up to 3 (such as any of 1 , 2, or 3) amino acid substitutions, the CDR2 comprises the amino acid sequence of SEQ ID N0:l 3 or a variant thereof comprising up to 3 (such as any of 1, 2, or 3) amino acid substitutions, and the CDR3 comprises the amino acid sequence of SEQ ID N0:21 or a variant thereof comprising up to 3 (such as any of 1 , 2, or 3) amino acid substitutions,- the C DR1 comprises the amino acid sequence of SEQ ID N 0:6 or a variant thereof comprising up to 3 (such as any of 1 , 2, or 3) amino acid substitutions, the CDR2 comprises the amino acid sequence of SEQ ID N0:l 4 or a variant thereof comprising up to 3 (such as any of 1, 2, or 3) amino acid substitutions, and the CDR3 comprises the amino acid sequence of SEQ ID N0:22 or a variant thereof comprising up to 3 (such as any of 1 , 2, or 3) amino acid substitutions; the C DR1 comprises the amino acid sequence of SEQ ID N 0: 7 or a variant thereof comprising up to 3 (such as any of 1 , 2, or 3) amino acid substitutions, the CDR2 comprises the amino acid sequence of SEQ ID N 0:15 or a variant thereof comprising up to 3 (such as any of 1, 2, or 3) amino acid substitutions, and the CDR3 comprises the amino acid sequence of SEQ ID NO:23 or a variant thereof comprising up to 3 (such as any of 1 , 2, or 3) amino acid substitutions; or the C DR1 comprises the amino acid sequence of SEQ ID N 0:8 or a variant thereof comprising up to 3 (such as any of 1 , 2, or 3) amino acid substitutions, the CDR2 comprises the amino acid sequence of SEQ ID N0:l 6 or a variant thereof comprising up to 3 (such as any of 1, 2, or 3) amino acid substitutions, and the CDR3 comprises the amino acid sequence of SEQ ID N0:24 or a variant thereof comprising up to 3 (such as any of 1 , 2, or 3) amino acid substitutions.

6. The isolated anti-FAP construct of any prior claim, wherein: the CDR1 comprises the amino acid sequence of SEQ ID N0:l, the CDR2 comprises the amino acid sequence of SEQ ID N0:9, and the CDR3 comprises the amino acid sequence of SEQ ID NO :17; the CDR1 comprises the amino acid sequence of SEQ ID N0:2, the CDR2 comprises the amino acid sequence of SEQ ID N0:l 0, and the CDR3 comprises the amino acid sequence of SEQ ID N0:l 8; the CDR1 comprises the amino acid sequence of SEQ ID N0:3, the CDR2 comprises the amino acid sequence of SEQ ID N0:l 1, and the CDR3 comprises the amino acid sequence of SEQ ID N0:l 9; the CDR1 comprises the amino acid sequence of SEQ ID N0:4, the CDR2 comprises the amino acid sequence of SEQ ID N0:l 2, and the CDR3 comprises the amino acid sequence of SEQ ID N0:20; the CDR1 comprises the amino acid sequence of SEQ ID N0:5, the CDR2 comprises the amino acid sequence of SEQ ID N0:l 3, and the CDR3 comprises the amino acid sequence of SEQ ID N0:21; the CDR1 comprises the amino acid sequence of SEQ ID NO:6, the CDR2 comprises the amino acid sequence of SEQ ID NO:14, and the CDR3 comprises the amino acid sequence of SEQ ID N0:22; the CDR1 comprises the amino acid sequence of SEQ ID NO:7, the CDR2 comprises the amino acid sequence of SEQ ID NO:15, and the CDR3 comprises the amino acid sequence of SEQ ID N0:23; or the CDR1 comprises the amino acid sequence of SEQ ID NO:8, the CDR2 comprises the amino acid sequence of SEQ ID NO:16, and the CDR3 comprises the amino acid sequence of SEQ ID N0:24.

1. The isolated anti-FAP construct of any prior claim, wherein the sd Ab moiety comprises any one of the following: a VHH domain comprising the amino acid sequence of SEQ ID NO:57 or a variant thereof having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID N0:57, wherein the CDR1 comprises the amino acid sequence of SEQ ID N0:l , the CDR2 comprises the amino acid sequence of SEQ ID NO :9, and the CDR3 comprises the amino acid sequence of SEQ ID N 0:17; a VHH domain comprising the amino acid sequence of SEQ ID NO:59 or a variant thereof having at least 90%, at least

95%, or at least 99% sequence identity to SEQ ID N 0 :59, wherein the CDR 1 comprises the amino acid sequence of SEQ ID NO :2, the

CDR2 comprises the amino acid sequence of SEQ ID NO:10, and the CDR3 comprises the amino acid sequence of SEQ ID N0:l 8; a VHH domain comprising the amino acid sequence of SEQ ID NO:61 or a variant thereof having at least 90%, at least

95%, or at least 99% sequence identity to SEQ ID N0:6l, wherein the CDR1 comprises the amino acid sequence of SEQ ID N0:3, the

CDR2 comprises the amino acid sequence of SEQ ID NO:11 , and the CDR3 comprises the amino acid sequence of SEQ ID N0:l 9; a VHH domain comprising the amino acid sequence of SEQ ID NO:63 or a variant thereof having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID N 0 :63, wherein the CDR 1 comprises the amino acid sequence of SEQ ID NO :4, the CDR2 comprises the amino acid sequence of SEQ ID NO:12, and the CDR3 comprises the amino acid sequence of SEQ ID N0:20; a VHH domain comprising the amino acid sequence of SEQ ID NO:65 or a variant thereof having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID N 0 :65, wherein the CDR 1 comprises the amino acid sequence of SEQ ID NO :5, the CDR2 comprises the amino acid sequence of SEQ ID NO:13, and the CDR3 comprises the amino acid sequence of SEQ ID N0:21 ; a VHH domain comprising the amino acid sequence of SEQ ID NO:67 or a variant thereof having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID N 0 :67, wherein the CDR 1 comprises the amino acid sequence of SEQ ID NO :6, the CDR2 comprises the amino acid sequence of SEQ ID NO:14, and the CDR3 comprises the amino acid sequence of SEQ ID N0:22; a VHH domain comprising the amino acid sequence of SEQ ID NO:69 or a variant thereof having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID N 0 :69, wherein the CDR 1 comprises the amino acid sequence of SEQ ID NO :7, the CDR2 comprises the amino acid sequence of SEQ ID NO :15, and the C DR3 comprises the amino acid sequence of SEQ ID N 0 :23; or a VHH domain comprising the amino acid sequence of SEQ ID NO:71 or a variant thereof having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID N0:7l, wherein the CDR1 comprises the amino acid sequence of SEQ ID N0:8, the CDR2 comprises the amino acid sequence of SEQ ID NO:16, and the CDR3 comprises the amino acid sequence of SEQ ID N0:24.

8. The isolated anti-FAP construct of any prior claim, wherein the sdAb moiety specifically recognizing FAP is camelid, chimeric, partially humanized, or fully humanized.

9. The isolated anti-FAP construct of any prior claim, wherein the sd Ab moiety specifically recognizing FAP is fused to a human IgGl Fc.

10. The isolated anti-FAP construct of any prior claim, wherein the isolated anti-FAP construct is a heavy chain-only antibody.

11. The isolated anti-FAP construct of any prior claim, wherein the isolated anti-FAP construct is fused to a second antibody moiety.

12. The isolated anti-FAP construct of any prior claim, comprising two of the sd Ab moiety specifically recognizing FAP connected by a peptide linker.

13. The isolated anti-FAP construct of any prior claim, wherein the isolated anti-FAP construct is labeled.

14. The isolated anti-FAP construct of any prior claim, wherein the isolated anti-FAP construct is radiolabeled.

15. A pharmaceutical composition comprising the isolated anti-FAP construct of any prior claim and a pharmaceutically acceptable carrier.

16. A method of treating a FAP-related disease in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of the pharmaceutical composition of claim 15.

17. The method of claim 16, wherein the FAP-related disease is cancer.

18. The method of claim 17, wherein the cancer is a solid tumor.

19. The method of any one of claims 16-18, further comprising screening the individual, comprising administering a screening amount of the pharmaceutical composition to the individual and imaging the individual for presence of the isolated anti-

FAP construct in the individual.

20. A method of screening an individual, comprising administering a screening amount of the pharmaceutical composition of claim 15 to the individual and imaging the individual for presence of the isolated anti-FAP construct in the individual.