constrained conditionally activated binding protein

Abstract

The present invention relates to conditional bispecific redirected activation constructs or COBRAs administered in the form of active prodrugs. Upon exposure to tumor proteases, the constructs are cleaved and activated so that they can bind both tumor target antigens (TTA) and CD3, thereby recruiting CD3-expressing T cells to the tumor for treatment. In some embodiments, the tumor target antigen is B7H3.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 814,210, filed March 5, 2019; U.S. Provisional Application No. 62 / 814,744, filed March 6, 2019; and U.S. Provisional Application No. 62 / 826,523, filed March 29, 2019, the disclosures of which are incorporated herein by reference in their entirety. Background of the Invention

[0004] In various clinical settings, there is often a need to selectively destroy individual cells or specific cell types. For example, a primary goal of cancer therapy is to specifically destroy tumor cells while leaving healthy cells and tissues as intact as possible. One such approach is to induce an immune response against the tumor, causing immune effector cells such as natural killer (NK) cells or cytotoxic T lymphocytes (CTLs) to attack and destroy tumor cells.

[0005] The use of intact monoclonal antibodies (mAbs), which offer excellent binding specificity and affinity for tumor-associated antigens, has been successfully applied in cancer treatment and diagnosis. However, the large size of intact mAbs, their poor biodistribution, low potency, and persistence in the blood pool limit their clinical application. For example, intact antibodies can exhibit specific accumulation within tumor regions. In biodistribution studies, when tumors are examined precisely, a heterogeneous distribution of antibodies with primitive accumulation is observed in peripheral areas. Due to tumor necrosis, heterogeneous antigen distribution, and increased stromal pressure, it is impossible to reach the central portion of the tumor with intact antibody constructs. In contrast, smaller antibody fragments exhibit rapid tumor localization, deeper penetration into the tumor, and relatively rapid clearance from the bloodstream. However, many antibodies (including scFv and other constructs) exhibit an "on target / off tumor" effect, where the molecule is active on non-tumor cells, causing side effects, some of which may be toxic. This invention relates to novel constructs that are selectively activated in the presence of tumor proteases. Summary of the Invention

[0006] This invention provides a variety of different protein compositions for treating cancer. Therefore, in one aspect, this invention provides a “form 2” protein comprising, from the N-terminus to the C-terminus: a) a first single-domain antigen-binding domain (sdABD) (sdABD-TTA) binding to a human tumor target antigen (TTA); b) a domain linker; c) a constrained Fv domain comprising: i) a variable heavy domain comprising vhCDR1, vhCDR2, and vhCDR3; ii) a constrained non-cleavable linker (CNCL); and iii) a variable light domain comprising vlCDR1, vlCDR2, and vlCDR3; d) a second domain linker; e) a second sdABD. -TTA; f) a cleavable linker (CL); g) a constrained pseudo-Fv domain comprising: i) a pseudo-light variable domain; ii) a non-cleavable linker (NCL); and iii) a pseudo-heavy variable domain; h) a third domain linker; and i) a third sdABD bound to human serum albumin; wherein the variable heavy domain and the variable light domain are capable of binding human CD3, but the constrained Fv domain does not bind CD3; the variable heavy domain and the pseudo-variable light domain intramolecularly associate to form an inactive Fv; and the variable light domain and the pseudo-variable heavy domain intramolecularly associate to form an inactive Fv. In some embodiments, the human tumor target antigen is B7H3.

[0007] In another aspect, the present invention provides a protein comprising, from its N-terminus to its C-terminus: a) a first single-domain antigen-binding domain (sdABD) (sdABD-TTA) binding to a human tumor target antigen (TTA), the first single-domain antigen-binding domain comprising sdFR1-sdCDR1-sdFR2-sdCDR2-sdFR3-sdCDR3-sdFR4; b) a first domain linker; and c) a constrained Fv domain comprising: i) comprising ii) a variable heavy structural domain containing vlFR1-vlCDR1-vlFR2-vlCDR2-vlFR3-vlCDR3-vlFR4; and iii) a constrained, non-disintegrable joint (CNCL); d) a second structural domain joint; e) a second sdABD-TTA; f) a disintegrable joint (CL); and g) a constrained pseudo-Fv structural domain, wherein... The constrained pseudo-Fv domain comprises: i) a pseudo-light variable domain comprising sdFR1-sdCDR1-sdFR2-sdCDR2-sdFR3-sdCDR3-sdFR4; ii) a non-disintegrable linker (NCL); and iii) a pseudo-heavy variable domain comprising vlFR1-vlCDR1-vlFR2-vlCDR2-vlFR3-vlCDR3-vlFR4; h) a third domain linker; and i) binding to human blood. The third sdABD of albumin, comprising sdFR1-sdCDR1-sdFR2-sdCDR2-sdFR3-sdCDR3-sdFR4; wherein the variable heavy domain and the variable light domain are capable of binding human CD3, but the constrained Fv domain does not bind CD3; the variable heavy domain and the pseudo-variable light domain intramolecularly associate to form an inactive Fv; and the variable light domain and the pseudo-variable heavy domain intramolecularly associate to form an inactive Fv. In some embodiments, the human tumor target antigen is B7H3.

[0008] In some embodiments of the form 2 protein, the variable heavy domain is located at the N-terminus of the variable light domain, and the pseudo-light variable domain is located at the N-terminus of the pseudo-variable heavy domain. In some embodiments, the variable heavy domain is located at the N-terminus of the variable light domain, and the pseudo-variable light domain is located at the C-terminus of the pseudo-variable heavy domain. In some embodiments, the variable heavy domain is located at the C-terminus of the variable light domain, and the pseudo-variable light domain is located at the N-terminus of the pseudo-variable heavy domain. In some embodiments, the variable heavy domain is located at the C-terminus of the variable light domain, and the pseudo-variable light domain is located at the C-terminus of the pseudo-variable heavy domain.

[0009] In some embodiments of the form 2 protein, the first sdABDTTA and the second sdABDTTA are the same. In some embodiments, the first sdABDTTA and the second sdABDTTA are different. In these implementations, sdABD-TTA is selected from those depicted in Figure 5, including SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 53, SEQ ID NO: 57, SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 69, SEQ ID NO: 73, 77, SEQ ID NO: 81, SEQ ID NO: 85, SEQ ID NO: 89, SEQ ID NO: 93, SEQ ID NO: 97, SEQ ID NO: 101, SEQ ID NO: 105, SEQ ID NO: 109 and SEQ ID NO: 113.

[0010] In some embodiments of the form 2 protein, the pseudo-revariable domain of the constrained pseudo-Fv domain is selected from SEQ ID NO: 146(V Hi ), SEQ ID NO: 150 (V Hi2 The group consisting of SEQ ID NO: 154 (VHiGL4) is shown in Figure 5. In some embodiments, the pseudo-light variable domain of the constrained pseudo-Fv domain is selected from SEQ ID NO: 130 (VHiGL4). Li ), SEQ ID NO: 134(V Li2) and SEQ ID NO: 138(V LiGL The group is shown in Figure 5.

[0011] In another aspect, the present invention provides a "Form 1" protein comprising, from its N-terminus to its C-terminus: a) a first sdABD-TTA; b) a first domain linker; c) a constrained Fv domain comprising: i) a first variable heavy domain comprising vhCDR1, vhCDR2, and vhCDR3; ii) a constrained cleavable linker (CCL); and iii) a first variable light domain comprising vlCDR1, vlCDR2, and vlCDR3; d) a second domain linker; e) a second sdABD-TTA; f) a cleavable linker (CL); g) a constrained pseudo- The constrained pseudo-Fv domain comprises: i) a first pseudo-light variable domain; ii) a non-cleavable linker (NCL); and iii) a first pseudo-heavy variable domain; h) a third domain linker; and i) a third sdABD bound to human serum albumin; wherein the first variable heavy domain and the first variable light domain are capable of binding human CD3, but the constrained Fv domain does not bind CD3; wherein the first variable heavy domain and the first pseudo-variable light domain are intramolecularly associated to form an inactive Fv; and wherein the first variable light domain and the first pseudo-variable heavy domain are intramolecularly associated to form an inactive Fv. In another aspect, the present invention provides a "Form 4" protein comprising, from its N-terminus to its C-terminus: a) a first single-domain antigen-binding domain (sdABD) (sdABD-TTA) that binds to a human tumor target antigen (TTA); b) a first domain linker; c) a constrained Fv domain comprising: i) a first variable heavy domain comprising vhCDR1, vhCDR2, and vhCDR3; ii) a constrained non-cleavable linker (CNCL); and iii) a first variable light domain comprising vlCDR1, vlCDR2, and vlCDR3; d) a cleavable linker (CL). e) a second sdABD bound to human serum albumin; f) a domain linker; g) a constrained pseudo-Fv domain comprising: i) a first pseudo-light variable domain; ii) a non-cleavable linker (NCL); and iii) a first pseudo-heavy variable domain; wherein the first variable heavy domain and the first variable light domain are capable of binding human CD3, but the constrained Fv domain does not bind CD3; wherein the first variable heavy domain and the first pseudo-variable light domain intramolecularly associate to form an inactive Fv; and the first variable light domain and the first pseudo-variable heavy domain intramolecularly associate to form an inactive Fv.

[0012] In another aspect of the proteins of forms 1, 2, and 4 listed above, the first variable heavy structure domain is located at the N-terminus of the first variable light structure domain, and the pseudo-light variable structure domain is located at the N-terminus of the pseudo-variable heavy structure domain.

[0013] In another aspect of the proteins of forms 1, 2, and 4 listed above, the first variable heavy structure domain is located at the N-terminus of the first variable light structure domain, and the pseudo variable heavy structure domain is located at the N-terminus of the pseudo variable light structure domain.

[0014] In another aspect of the proteins of forms 1, 2, and 4 listed above, the first variable light domain is located at the N-terminus of the first variable heavy domain, and the pseudo-light variable domain is located at the N-terminus of the pseudo-variable heavy domain.

[0015] In another aspect of the proteins of forms 1, 2, and 4 listed above, the first variable light domain is located at the N-terminus of the first variable heavy domain, and the pseudo-variable heavy domain is located at the N-terminus of the pseudo-variable light domain.

[0016] In another aspect, the present invention provides form 1 and form 2 proteins, wherein the first and second TTAs are identical.

[0017] In another respect, the present invention provides form 1 and form 2 proteins, wherein the first and second TTAs are different.

[0018] In another aspect, the present invention provides proteins of form 1, form 2 and form 4, wherein the first and second TTAs are selected from EGFR, EpCAM, FOLR1, Trop2, ca9 and B7H3. These sequences can be selected from the following groups: SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 53, SEQ ID NO: 57, SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 69, SEQ ID NO: 73, 77, SEQ ID NO: 81, SEQ ID NO: 85, SEQ ID NO: 89, SEQ ID NO: 93, SEQ ID NO: 97, SEQ ID NO: 101, SEQ ID NO: 105, SEQ ID NO: 109 and SEQ ID NO: 113.

[0019] In another aspect, the present invention provides proteins of form 1, form 2 and form 4, wherein the half-life extension domain has SEQ ID NO: 117 (aHSA(10GE)) and SEQ ID NO: 121 (aHSA with His tag).

[0020] In another aspect, the present invention provides proteins of forms 1, 2, and 4, wherein the cleavable linker is cleaved by human proteases selected from the group consisting of: MMP2, MMP9, Meprin A, Meprin B, cathepsin S, cathepsin K, cathepsin L, granzyme B, uPA, Kallekriein 7, proteolytic enzymes, and thrombin, or other human proteases as described in FIG6.

[0021] In another aspect, the present invention provides a protein selected from the group consisting of: Pro186, Pro225, Pro226, Pro233, Pro262, Pro311, Pro312, Pro313, Pro356, Pro359, Pro364, Pro388, Pro448, Pro449, Pro450, Pro451, Pro495, Pro246, Pro254, Pro255, Pro256, Pro420, Pro421, Pro432, Pro479, Pro480, Pro187, Pro221, Pro222, Pro223, Pro224, Pro393, Pro394, Pro395, Pro396, Pro429, Pro430, Pro431, Pro601, Pro602, V3 and V4, Pro664, Pro6 65, Pro667, Pro694, Pro695, Pro565, Pro566, Pro567, Pro727, Pro728, Pro729, Pro730, Pro731, Pro676, Pro677, Pro678, Pro679, Pro808, Pro819, Pro621, Pro622, Pro640, Pro641, Pro642, Pro643, Pro744, Pro746, Pro638, Pro639, Pro396, Pro476, Pro706, Pro709, Pro470, Pro471, Pro551, Pro552, Pro623, Pro624, Pro698, Pro655, Pro656, Pro657, Pro658, Pro516, Pro517, Pro518 and Pro519.

[0022] In another aspect, the present invention provides nucleic acids encoding proteins of form 1, form 2 or form 4 as described herein, as well as expression vectors and host cells comprising nucleic acids encoding said proteins.

[0023] In another aspect, the present invention provides a method for preparing the protein of the present invention and a method for treating patients in need.

[0024] In another aspect, the present invention provides a composition comprising a "form 3A" prodrug protein pair, the composition comprising: a) a first protein comprising, from the N-terminus to the C-terminus: i) a first sdABD-TTA; ii) a first domain linker; iii) a pseudo-Fv domain comprising, from the N-terminus to the C-terminus: 1) a variable heavy chain comprising vhCDR1, vhCDR2, and vhCDR3; 2) a cleavable linker; 3) a first pseudo-variable light domain comprising iVLCDR1, iVLCDR2, and iVLCDR3; iv) a second domain linker; v) sdABD-HSA; a) a second protein comprising, from the N-terminus to the C-terminus: i) a third sdABD bound to a human tumor target antigen; ii) a third domain. iii) a pseudo-Fv domain, the pseudo-Fv domain comprising, from the N-terminus to the C-terminus: 1) a variable light chain comprising VLCDR1, VLCDR2, and VLCDR3; 2) a cleavable linker; and 3) a first pseudo-variable heavy structure domain comprising iVHCDR1, iVHCDR2, and iVHCDR3; iv) a fourth domain linker; v) sdABD-HSA; wherein the first variable heavy structure domain and the first variable light structure domain are capable of binding human CD3 upon association; wherein the first variable heavy structure domain and the first pseudo-variable light structure domain intermolecularly associate to form an inactive Fv; wherein the first variable light structure domain and the first pseudo-variable heavy structure domain intermolecularly associate to form an inactive Fv; and wherein the first and third sdABDs are selected from the group consisting of: SEQ SEQ ID NO: 17 ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 69, SEQ ID NO: 73,77, SEQ ID NO: 81, SEQ ID NO: 85, SEQ ID NO: 89, SEQ ID NO: 93, SEQ ID NO: 97, SEQ ID NO: 101, SEQ ID NO: 105, SEQ ID NO: 109 and SEQ ID NO: 113.

[0025] In another aspect, the present invention provides a composition comprising a "form 3B" prodrug protein pair, the composition comprising a) a first protein comprising, from the N-terminus to the C-terminus: i) a first sdABD-TTA; ii) a first domain linker; iii) a second sdABD-TTA; iv) a second domain linker; iii) a pseudo-Fv domain comprising, from the N-terminus to the C-terminus: 1) a variable heavy chain comprising vhCDR1, vhCDR2, and vhCDR3; 2) a cleavable linker; 3) a first pseudo-variable light domain comprising iVLCDR1, iVLCDR2, and iVLCDR3; iv) a third domain linker; and v) sdABD-HSA; and a) a first second protein comprising, from the N-terminus to the C-terminus: i) a third sdABD-TTA; ii) a first domain linker; iii) a second domain linker; iv) a third domain linker; and v) sdABD-HSA; and a third second protein comprising, from the N-terminus to the C-terminus: i) a third sdABD-TTA; ii) a first domain linker; iii) a second domain linker; iv) a third domain linker; and v ... -TTA; ii) fourth domain connector; iii) fourth sdABD-TTA; iv) fifth domain connector; iii) pseudo-Fv domain, which from the N-terminus to the C-terminus comprises: 1) a variable light chain comprising VLCDR1, VLCDR2 and VLCDR3; 2) a cleavable connector; and 3) a first pseudo-variable heavy domain comprising iVHCDR1, iVHCDR2 and iVHCDR3; iv) a sixth domain connector; v) sdABD-HSA; wherein the first variable heavy domain and the first variable light domain are capable of binding human CD3 upon association; wherein the first variable heavy domain and the first pseudo-variable light domain intermolecularly associate to form an inactive Fv; and wherein the first variable light domain and the first pseudo-variable heavy domain intermolecularly associate to form an inactive Fv.

[0026] On the other hand, the form 3A and form 3B proteins have sdABD-HSA, which has SEQ ID NO: 117 or SEQ ID NO: 121.

[0027] On the other hand, form 3A and form 3B proteins have sdABD-TTA that binds to TTAs selected from EGFR, EpCAM, Trop2, CA9, FOLR1, and B7H3. The sdABD-TTA can be selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 53, SEQ ID NO: 57, SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 69, SEQ ID NO: 73, 77, SEQ ID NO: 81, SEQ ID NO: 85, SEQ ID NO: 89, SEQ ID NO: 93, SEQ ID NO: 97, SEQ ID NO: 101, SEQ ID NO: 105, SEQ ID NO: 109 and SEQ ID NO: 113.

[0028] In another aspect, the present invention provides an sdABD that binds to human Trop2, the sdABD having a sequence selected from SEQ ID NO: 77, SEQ ID NO: 81, SEQ ID NO: 85, SEQ ID NO: 89 and SEQ ID NO: 93.

[0029] In another aspect, the present invention provides an sdABD that binds to human B7H3, the sdABD having a sequence selected from SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 53 and SEQ ID NO: 57.

[0030] In another aspect, the present invention provides an sdABD that binds to human CA9, the sdABD having a sequence selected from SEQ ID NO: 101, SEQ ID NO: 105, SEQ ID NO: 109 and SEQ ID NO: 113.

[0031] In another aspect, the present invention provides an sdABD that binds to human EpCAM, the sdABD having a sequence selected from SEQ ID NO: 69 and SEQ ID NO: 73.

[0032] In another aspect, the present invention provides a nucleic acid composition comprising a first nucleic acid encoding a first protein member of a prodrug pair and a second nucleic acid encoding a second protein member of the pair; and an expression vector and a host cell containing the nucleic acid. Attached Figure Description

[0033] Figure 1 The “Form 1” type of protease activation described in this invention is referred to herein as a “constrained, non-cleavable construct” or “cc construct.” In this embodiment, a representative construct is Pro140: two TTAs are present in ABD (e.g., Figure 1 (These are all described as identical, although they may be different as described herein). Upon cleavage, the prodrug construct splits into three components: an α-TTA domain containing an active VH linked to αCD3 via a domain linker; a second α-TTA domain containing an active VL linked to αCD3 via a domain linker; and a “remaining” portion containing a half-life-extending domain linked to inactive VH and VL. The two active variable domains then freely associate to form a functional anti-CD3 binding domain. It should be noted that in the “Form 1” embodiment, the resulting active component is trivalent: it exhibits monovalent binding to CD3 and divalent binding to TTA, thereby producing a bispecific binding protein, although in some cases this trivalent binding may be trispecific, exhibiting monovalent binding to CD3, monovalent binding to a first TTA, and monovalent binding to a second TTA. Figure 1 The anti-human serum albumin (HSA) domain, which is also shown as a half-life extension domain, is described in many embodiments as sdABD as defined herein, although this is optional and / or may be replaced by other half-life extension domains as described herein; in addition, the half-life extension domain may also be located at the N-terminus or inside the construct. Figure 1 There are also Fvs with VH and VL in a specific order, and pseudo-Fvs with iVH and iVL, for example, from the N-terminus to the C-terminus, VH-connector-VL (and iVL-connector-iVH), although these can be reversed (VL-connector-VH and iVH-connector-iVL), as those skilled in the art will recognize. Alternatively, one of these Fvs may be in one orientation and the other in another orientation, although the protein in the orientation shown here is unexpectedly expressed more than the other orientations.

[0034] Figure 2The “Form 2” type of protease activation described herein is referred to herein as a “constrained, non-cleavable construct” or “CNCL construct,” and is sometimes also referred to herein as a “dimerized construct” as discussed herein. These constructs are not isomerized as discussed herein. Upon cleavage, the two prodrug constructs split into four components: two half-life-extending domains (in this case, sdABD to HSA) linked to two pseudodomains (which may or may not self-associate depending on the linker length and inactivation mutation), and two active portions that self-assemble into a dimer active moiety containing four anti-TTA domains (which may be all identical or two identical and the other two different). It should be noted that in the “Form 2” embodiment, the resulting active component is hexavalent: exhibiting bivalent binding to CD3 and tetravalent binding to TTA, thus producing a bispecific binding protein, although in some cases this hexavalent binding may be trispecific, having bivalent binding to CD3, bivalent binding to a first TTA, and bivalent binding to a second TTA. Figure 2 The anti-human serum albumin (HSA) domain, which is also shown as a half-life extension domain, is described in many embodiments as sdABD as defined herein, although this is optional and / or may be replaced by other half-life extension domains as described herein; in addition, the half-life extension domain may also be located at the N-terminus or inside the construct. Figure 2 There are also Fvs with VH and VL in a specific order, and pseudo-Fvs with iVH and iVL, for example, from the N-terminus to the C-terminus, VH-connector-VL (and iVL-connector-iVH), although these can be reversed (VL-connector-VH and iVH-connector-iVL), as those skilled in the art will recognize. Alternatively, one of these Fvs may be in one orientation and the other in another orientation, although the protein in the orientation shown here is unexpectedly expressed more than the other orientations.

[0035] Figures 3A-3B Constructs of the “Form 3” type as described in this article are sometimes also called “semi-constructs” or “semi-COBRAs”. TM"This is because these are two distinct polypeptide chains that together constitute the MCE therapeutic agent, as further discussed herein. In this embodiment, the constructs are delivered in pairs, having pre-cleaved intramolecular self-assembly to produce an inactive anti-CD3 Fv domain. Upon cleavage, the inert variable domain is released, and the two active variable domains then intermolecularly assemble to form an active anti-CD3 binding domain. Both sdABD-TTAs bind to their respective receptors on the surface of tumor cells, and the cleavage is carried out via a protease. This allows for intermolecular assembly because the molecules are physically held in place, thus facilitating the assembly of the active anti-CD3 domain. As described above for forms 1 and 2, in this embodiment, the N-terminal to C-terminal order of the variable domains may also be reversed or mixed. Furthermore, sdABD(HSA) may be located at the N-terminus or C-terminus of each half-construct. Pro16 has sdABD(HSA) at the C-terminus, and Pro17 has it at the N-terminus (see Pro19, SEQ ID NO: XX, has sdABD(HSA) at the C-terminus). Figure 3A The diagram shows a Form 3 construct where each semi-construct has a single sdABD-TTA structural domain, and... Figure 3B Each semi-construct shown, in either "dual-targeting" or "heterogeneous-targeting" form, has two sdABD-TTA form 3 constructs. Note that... Figure 3B FORR1 and EGFR are used as two TTAs, but other combinations as outlined in this article may also be used.

[0036] Figure 4 A “Form 4” type construct is depicted, similar to the “Form 2” construct but containing only a single sdABD-TTA. The diagram illustrates sdABD-TTA targeting EGFR, but other TTAs may be used, as those skilled in the art will understand. Upon cleavage, the prodrug construct splits into two components: a half-life-extending domain linked to a pseudo-Fv (in this case, sdABD targeting HSA) and an active moiety, which self-assembles in the presence of a second active moiety from a different cleavage molecule into a dimerized active moiety containing two anti-TTA domains. It should be noted that in the “Form 4” embodiment, the resulting active component is tetravalent: it exhibits bivalent binding to CD3 and bivalent binding to TTA, resulting in a bispecific binding protein. Figure 4 The anti-human serum albumin (HSA) domain is also shown as a half-life extension domain, in many embodiments as sdABD(1 / 2) as defined herein, although as described herein this is optional and / or may be replaced by other half-life extension domains; additionally, the half-life extension domain may also be located at the N-terminus or inside the construct. Figure 4There are also Fvs with VH and VL in a specific order, and pseudo-Fvs with iVH and iVL, for example, from the N-terminus to the C-terminus, VH-connector-VL (and iVL-connector-iVH), although these can be reversed (VL-connector-VH and iVH-connector-iVL), as those skilled in the art will recognize. Alternatively, one of these Fvs may be in one orientation and the other in another orientation, although the protein in the orientation shown here is unexpectedly expressed more than the other orientations.

[0037] Figures 5A-5J The various sequences of the present invention are depicted. For antigen-binding domains, CDRs are underlined. As summarized more fully herein, these domains can be assembled in a wide range of configurations in the present invention, including “Form 1”, “Form 2”, “Form 3”, and “Form 4” orientations.

[0038] Figures 6A-6D Many suitable protease cleavage sites are depicted. As those skilled in the art will understand, these cleavage sites can be used as cleavable linkers. In some embodiments, for example, when a more flexible cleavable linker is required, additional amino acids (typically glycine and serine) may be present at the N-terminus or C-terminus or both of these cleavage sites.

[0039] Figures 7A-7D Depicting with "Form 3" or "Half-COBRA" TM "Some structural data. This indicates that the form 3 construct co-binds to CD3 and generates a CD3 binding site after being cleaved by a protease (in this case, an EK protease, but any protease cleavage site outlined herein and depicted in Figures 5 and 6) as shown by sandwich FACS analysis."

[0040] Figures 8A-8D This demonstrates that protease cleavage occurs via complementary hemi-cobra TM T cell killing of EGFR+ target cells in synergistic activation. Figure 8A and Figure 8B The isolated constructs, but cleaved with different concentrations of protease, did not affect target cell viability. However, Figure 8C The results showed that, in combination, the viability of target cells was significantly reduced in the presence of proteases. Figure 8D The general mechanism is shown.

[0041] Figure 9 Some non-target controls are shown for determining the efficacy of the construct in test form 1.

[0042] Figures 10A-10FThe generation of the active CD3-binding domain is shown to depend on target binding to two "arms" (e.g., the sdABD-TTA domain), one of which is present on each of the two constructs. ELISA assays are performed as described in the examples.

[0043] Figure 11 Show suitable half-cobra TM The diagram is correct. “Mep” represents the meprin protease cleavage site, “His-6” is a label as discussed more fully in this article, ST14 is the protein lyase protease cleavage site, and “Thb” is the thrombin protease cleavage site.

[0044] Figures 12A-12C Showing with Figure 11 TDCC data related to the build. Figure 12A This demonstrates the efficacy of adding pre-lysed hemi-COBRA in producing OvCAR8 cells. Figure 12B The addition of pre-lysed hemi-COBRA demonstrates its efficacy in producing HCT116 cells, and Figure 12C The results show that the addition of pre-lysicated hemi-COBRA produces efficacy against LoVo cells, all of which are cancer cell lines.

[0045] Figures 13A-13B The MMP9 linker was shown to be stable in vivo. NSG mice were administered a single intravenous bolus dose of Pro40 (MMP9 cleavable) and Pro74 (non-cleavable) at a dose of 0.5 mg / kg via tail vein. Dosage solutions of each compound were prepared in a medium of 25 mM citrate, 75 mM L-arginine, 75 mM NaCl, and 4% sucrose at pH 7.0. Two blood samples were collected from each animal at pre-selected times: one at the start of the study via orbital or submandibular hemorrhage, and the other at the end time point via cardiac puncture. Blood collection time points were 0.083, 1, 6, 24, 72, and 168 hours. Plasma was prepared from each individual blood sample using K2EDTA tubes. Concentrations were determined using an MSD assay employing a monoclonal antibody specific for HSA sdABD and detected using the EGFR extracellular domain.

[0046] Figure 14 Depicted in Figure 15 The experiment described in the text uses form 3A hemi-cobra. TMSchematic diagram of the constructs. Pro51 is a positive control because it is "always on," so it forms active anti-CD3 Fv. Pro98 is a negative control because its sdABD targets chicken oocyte lysozyme, which is not expressed by tumors. Pro77 and Pro53 are prodrug form 3A pairs, using sdABD and MMP9 cleavage sites targeting EGFR. Pro74 and Pro72 are negative control form 3A pairs because they do not have cleavage sites.

[0047] Figure 15 As shown, using the protocol described in the examples, the Form 1 construct regressed tumors in vivo using two different tumor cell lines implanted in mice. The antitumor activity of the hemi-COBRA constructs (Pro77 and Pro53) depends on the inclusion of anti-EGFRsdABD and MMP9 cleavable linkers, as well as active anti-CD3 Fv.

[0048] Figure 16 A schematic diagram of the next-generation form is shown, namely a full-length construct with two pseudo-Fv domains (with a cleavable site between them), as generally described in US 2018 / 0134789, which is hereby incorporated by reference. However, as shown in the figure below, this first-generation full-length construct does not exhibit very good conditionability because it is isomerizeable to form active and inactive constructs.

[0049] Figure 17 As shown, the Form 3A build pair actually exhibits better conditionality than the Pro100 first-generation full-length build.

[0050] Figure 18 Describing another first-generation full-length construct, the construct in Figure 19 The test was conducted.

[0051] Figure 19 The first-generation constructs are shown to exhibit high activity, such as poor conditionality, even in their unfractured form.

[0052] Figure 20 The first-generation full-length construct is shown to exhibit two monomer peaks on SEC analysis.

[0053] Figure 21 A schematic diagram illustrating the reason for the non-cleavage activity is that the full-length first-generation construct isomerizes to form two conformations: one inactive because no active anti-CD3 Fv (“bivalent scFv”) is formed, and the other active in the absence of a protease, i.e., a “single-chain biantibody” type conformation. See PEDS 23(8):667-677 (2010).

[0054] Figure 22The results of TDCC assays, run at 37°C for 2 days, are shown using first-generation single-chain constructs. The results indicate that the un-cleaved constructs exhibit strong killing effects. These results lead to the generation of form 1 constructs.

[0055] Figures 23A-23G The Form 1 construct used in this invention is shown. As those skilled in the art will understand and as described herein, these are depicted as having an sdABD-EGFR targeting portion, but sdABD targeting other TTAs may be used.

[0056] Figure 24 The form 1 construct (Pro140 in this case) is shown to form a single isomer that is stable at 37°C.

[0057] Figure 25 As measured by Octet assay, the form 1 construct binds very little to human CD3 in its unlysected form. The top line is Pro120, the middle line is Pro51 (positive control), and the bottom line is Pro140 held at 4°C or 37°C for 3 days.

[0058] Figure 26 Similarly, the form 1 construct is described as having very low TDCC activity in its un-cleaved form.

[0059] Figure 27 The specific form 1 construct Pro140 used in in vivo testing is depicted, using sdABD-EGFR and MMP9 cleavage sites as the target portion.

[0060] Figures 28A-28B The tumor regression using the form 1 construct is shown.

[0061] Figure 29 Describing the cleavage sites in the constrained Fv reveals several distinct fragments: partially cleaved fragments and completely cleaved fragments. Surprisingly, the partially cleaved form is more reactive than the completely cleaved form, leading to the generation of form 2.

[0062] Figure 30 Several schematic diagrams of Form 2 are shown, all using the sdABD-EGFR targeting domain; however, as described herein and listed in the sequence, sdABD targeting other TTAs may be used. Pro51 and Pro201 are positive controls (in the active "half" configuration and the active dimer configuration, respectively), and Pro214 is a full-length negative control because it has no cleavage site.

[0063] Figure 31The TDCC activity of construct Pro187 (Form 2) using a meprin cleavage site is shown. In the TDCC assay, Pro187 with pre-cleavage addition showed 1200-fold higher activity than with uncleaved addition. Pre-cleaved Pro187 exhibited activity intermediate between the positive controls Pro51 and Pro201. Uncleaved Pro187 exhibited activity similar to Pro214, which lacks a protease-cleavable linker.

[0064] Figure 32 The TDCC activity of the form 2 construct Pro186 using n MMP9 cleavage sites is shown. In the TDCC assay, Pro186 with pre-cleavage addition showed 18-fold higher activity than with uncleaved addition. Pre-cleaved Pro186 exhibited activity intermediate between the positive controls Pro51 and Pro201. Uncleaved Pro186 showed higher activity than Pro214, which does not contain a protease-cleavable linker, likely due to MMP activity generated by cells during the 48-hour assay.

[0065] Figure 33 The Pro186 construct was depicted binding to cells with varying levels of the EGFR receptor, but not to CHO cells that do not express EGFR on their cell surface. Pro186 saturated cells expressing different levels of EGFR at similar COBRA concentrations.

[0066] Figure 34 Shown in Figure 35 A schematic diagram of the Form 2 construct used in in vivo studies, which uses the sdABD-EGFR targeting domain throughout.

[0067] Figure 35 As shown, the form 2 construct Pro186 is highly effective at both dose levels and outperforms the form 1 construct Pro140 at lower doses.

[0068] Figure 36 Numerous form 2 constructs based on Pro186 but with different protease cleavage sites are described. Although all these constructs use sdABD-EGFR for both targeting domains, other sdABDs targeting different TTAs can also be used, and they can be the same or different. That is, they can all be homogeneous (making sdABDs target the same TTA) or heterogeneous (making one sdABD target the first TTA and another sdABD target a different TTA).

[0069] Figure 37Schematic diagrams depicting different forms of constructions with varying linker lengths between Fv domains. These are illustrated using the MMP9 cleavage site, but other cleavage sites may also be used as described herein. Similarly, while all these constructions use sdABD-EGFR for both target domains, other sdABDs targeting different TTAs may also be used, and they may be the same or different.

[0070] Figure 38 The pseudo-Fv linker lengths can be varied; for example, construct 2 with short linkers between active Fvs (“short active”) and longer linkers between pseudo-Fvs (“long inactive”) exhibits similar activity to both “short active” and “short inactive” constructs. Therefore, the conditionality of the COBRA construct is independent of both constrained active and inactive scFv linkers; single-chain biantibody folding appears to be more advantageous than bivalent scFv folding as long as one of them is constrained.

[0071] Figure 39 The linker length of the active Fv can be varied; for example, the Form 2 construct with "long active" and "short inactive" exhibits similar behavior to the "short active" and "short inactive" constructs. Therefore, the conditionality of the COBRA construct is independent of both the constrained active and inactive scFv linkers; single-chain biantibody folding appears to be more favorable than bivalent scFv folding, provided one of them is constrained.

[0072] Figure 40A one Figure 40C Schematic diagrams of several different constructs are shown. Pro188 is a form 1 construct, similar to Pro140 but with a long connector (16-mer) in the pseudo-Fv. Pro189 and Pro190 (form 2 constructs) are similar to Pro186 and Pro187, except for having a long connector (16-mer) in the pseudo-Fv domain. Pro191 and Pro192 (also form 2 constructs) are similar to Pro189 and Pro190, except that they have an additional cleavage site upstream of sdABD(1 / 2). Pro193 (form 4) has a single EGFR targeting domain rearranged to iVH and iVL in reverse order, and an additional cleavage site upstream of sdABD(1 / 2). Pro195 is a form 2 construct similar to Pro186, with a targeting domain that binds to the same TTA (i.e., EGFR) but to a different epitope. Pro196, Pro197, and Pro198 are form 2 constructs with rearranged variable domains.

[0073] Figure 41The following facts are illustrated: different sdABD clones against human FOLR1 exhibit varying killing effects. The Pro22 construct (Pro51 with the FLAG sequence instead of NCL) binding to human FOLR1 was compared with Pro22-EGFR constructs targeting multiple cell line families.

[0074] Figure 42 Schematic diagrams depict four sdABD-FOLR1 constructs, including a Pro201 active domain dimer (two molecules intermolecularly associate to form two active Fvs against CD3) using sdABD-EGFR2 as a positive control, Pro311 using 77.2 sdABD and Pro312 using h59.3 sdABD, and two negative controls, Pro299 using h77.2 sdABD and Pro303 using h59.3 sdABD.

[0075] Figure 43 A schematic diagram depicting the Form 2 construct used in the FOLR / MMP9 in-body design.

[0076] Figure 44 The efficacy of the Pro312 construct in vivo was demonstrated, and the MMP9 cleavable linker was shown to be essential for antitumor activity.

[0077] Figure 45 Schematic diagrams depicting some forms of sdABD (sdABD-B7H3) targeting human B7H3, including Pro244, a positive control (using sdABD-B7H3 (hF7), and two form 2 test samples, Pro225 (form 2 construct) and Pro295 (negative control lacking cleavage site).

[0078] Figure 46 As shown, the Pro225 has greater conditionality compared to the control Pro295.

[0079] Figure 47 As shown, the Form 2 construct Pro373, using the meprin connector, exhibits significant conditionality compared to Pro295.

[0080] Figure 48 Numerous sdABD-B7H3 (using the hF12 sequence) constructs are depicted, showing the Pro51 positive control using sdABD-EGFR, the Pro244 positive control using sdABD-hF12 B7H3, the test construct Pro226, and the negative control Pro296 without cleavage sites.

[0081] Figure 49The good condition of the Pro226 construct in TDCC assays is shown.

[0082] Figure 50 This illustrates the humanization of sdABD for human EpCAM.

[0083] Figure 51 The diagrams show various forms: Pro244 is a positive control for the standard T-cell connector, Pro205 is a positive control for the active domain dimer, Pro199 is the form 2 construct, and Pro175 is a negative control.

[0084] Figure 52 The TDCC activity of the sdABD-EpCAM construct is shown, indicating good conditionality.

[0085] Figures 53A-53B The TDCC activity of the sdABD-EpCAM Pro199 construct was demonstrated, thus showing good conditionality in HT29 and LoVo cell models.

[0086] Figures 54A-54B The TDCC activity of the sdABD-EpCAM Pro200 construct was demonstrated, thus showing good conditionality in HT29 and LoVo cell models.

[0087] Figure 55 The diagram shows Pro255, which uses two different sdABD-TTAs compared to Pro199, which uses dual EpCAM sdABDs: one targeting EGFR (sdABD-EGFR) and the other targeting EpCAM (sdABD-EpCAM). These are sometimes referred to as “heterogeneous targeting” constructs in this paper, and in this case, as form 2 constructs.

[0088] Figure 56 As shown, the Pro255 dual-targeting molecule with the MMP9 cleavage site exhibits good conditionality.

[0089] Figures 57A-57D Results of experiments on three different cell types are shown. First, Raji transfectants with similar expression levels of EpCAM, EGFR, and EpCAM+EGFR were generated (data not shown). Then, Pro255 targeting both EpCAM and EGFR was tested in a TDCC analysis using each cell type. Figure 57A The parental Raji strain that does not express any receptor is shown. Figure 57B The conditionality of the EpCAM strain is shown. Figure 57C The conditionality of the EGRF strain is shown. Figure 57D The conditionality of the EpCAM / EGFR strain is shown.

[0090] Figure 58 A schematic diagram depicting the Form 4 constructor Pro258.

[0091] Figures 59A-59B The results show that Pro258 is conditional in both FBS and human serum. The conditionality of the MMP9 linker is underestimated due to MMP9 activity in the culture. Interestingly, Pro51 TDCC activity is inhibited by HSA binding, while Pro258 TDCC activity is similar to that of Pro51 in the presence of HSA. Finally, the conditionality of Pro258 is enhanced 6-fold in the presence of HSA.

[0092] Figures 60A-60C . Figure 60A This shows the cleavage of MMP9 substrates by other MMPs. Figure 60B and 60C The fragmentation of the FRET probe containing the MMP9 connector sequence is shown.

[0093] Figures 61A-61B Some exemplary constructs and their forms are shown.

[0094] Figures 62A-62V Many sequences of the present invention are shown, but many other sequences have been found in the sequence listing. CDRs are underlined and bolded, adapters are double-underlined (where cleavable adapters are italicized and double-underlined), and domain separators are indicated by " / ". All His6 tags are optional, as they can be used to reduce immunogenicity in humans and as purification tags.

[0095] Figures 63A to 63EE The amino acid sequence of an exemplary form 2 construct containing multiple sdABD-B7H3 and pseudo-Fv domains (e.g., Vli2 / Vhi2 domains) is described. Figure 63A Describe the amino acid sequences of Pro601 and Pro602. Figure 63B Describe the amino acid sequences of V3 and V4. Pro601 contains two identical sdAbs that bind B7H3 (e.g., aB7H3 hF7 sdAb). Pro602 contains two identical sdAbs that bind B7H3 (e.g., anti-B7H3 hF12 sdAb). V3 contains two different sdAbs that bind B7H3 (e.g., anti-B7H3 hF7 sdAb and anti-B7H3 hE12 sdAb). V4 contains two different sdAbs that bind B7H3 (e.g., anti-B7H3 hF7 sdAb and anti-B4H3 hF12 sdAb).

[0096] Figures 64A-64C The folding mechanism designed and predicted by COBRA is shown. Figure 64ADescribing Pro186 COBRA ( Figure 62B A schematic diagram of (SEQ ID NO: 145). Figure 64B The predicted COBRA folds are shown. Figure 64C The analytical size exclusion chromatogram of Pro186 is shown.

[0097] Figures 65A-65C Exemplary embodiments of the constructs described herein include Pro186 (pre-cleaved), Pro186 cleavage products, and PRO186 active dimers.

[0098] Figure 66 Provides instructions on how COBRA is converted into an active dimer after protease cleavage.

[0099] Figures 67A-67B Provides characterization of COBRA binding. Figure 67A The study demonstrated binding activity against human, cynomolgus monkey, and mouse products. Figure 67B The binding of PRO186 to human CD3ε is shown; the active PRO186 binding to human CD3ε and the active PRO186 binding to human EGFR are also shown.

[0100] Figures 68A-68B The cleavage of the PRO186 connector by MMP2 and MMP9 is shown. Figure 68A To depict the protein imprint of the active binding product molecules after cleavage. Figure 68B This shows the cumulative effect of active binding product molecules relative to cleavage time.

[0101] Figure 69 The in vitro activity of the conditional PRO186 construct is shown. Figure 69 - The left figure shows the results of the T cell killing assay. Figure 69 - The right figure shows the level of IFN-γ release in relation to the concentration of the test sample.

[0102] Figure 70 The relative activity of EGFR expression is shown in three tumor cell lines: LoVo (colorectal cancer (CRC) cell line), HT-29 (colorectal cancer (CRC) cell line), and SCC25 (head and neck cancer cell line).

[0103] Figure 71A and Figure 71B The expression of EGFR, MMP2, and MMP9 is shown in tumor cells and tumor xenografts. Figure 71A The surface density of EGFR cells is shown on three cancer cell lines—LoVo, HT-29, and SCC25. Figure 71B Immunohistochemical staining of EGFR, MMP2, and MMP9 in tumor xenografts is shown.

[0104] Figure 72 A schematic diagram of the experimental procedure for an adoptive human T-cell transfer model in tumor-bearing mice is provided.

[0105] Figure 73 The study demonstrates the regression of solid tumors established in mice by PRO186. Figure 73 - The left image shows the regression of a LoVo-derived tumor. Figure 73 - The middle image shows the regression of a tumor derived from HT-29. Figure 73 - The right figure shows the regression of tumors originating from SCC25.

[0106] Figures 74A-74B The results show that cleaved PRO186 is cleared faster than intact (uncleaved) PRO186. Figure 74A The pharmacokinetics of the test substance in the plasma of non-tumor-bearing mice are shown. Figure 74B The tumor volume of LoVo-derived tumors in mice that were administered the test sample is shown.

[0107] Figure 75 Results are depicted using lysed Pro233 with MMP9 and a T-cell-dependent cytotoxicity (TDCC) assay of Pro233. Pro233 is dependent on a humanized anti-EGFR binding domain. The results indicate that lysed Pro233 is potent in EGFR-expressing HT29 cells compared to the unlysed form.

[0108] Figure 76 The results of TDCC assays using Pro565, Pro565 cleaved by MMP9, and Pro568 (a non-cleaved control) are depicted. Pro565 is dependent on the hVIB664 anti-EpCAM binding domain. The results indicate that, on EpCAM-expressing HT29 tumor cells, cleaved Pro565 is more effective than the non-cleaved form and the non-cleaved control.

[0109] Figure 77 The results of TDCC assays using Pro225, Pro225 cleaved by MMP9, and Pro295 (a non-cleavable control) are depicted. Pro225 is dependent on the hF7 anti-B7H3 binding domain. The results indicate that, on B7H3-expressing HT29 tumor cells, cleaved Pro566 is more effective than the non-cleavable form and the non-cleavable control (note that HT29 expresses EGFR, B7H3, and EpCAM).

[0110] Figure 78The results of TDCC assays using Pro566, Pro566 cleaved by MMP9, and Pro569 (a non-cleaved control) are depicted. Pro566 is dependent on the hVIB665 anti-EpCAM binding domain. The results indicate that, on EpCAM-expressing HT29 tumor cells, cleaved Pro566 is more effective than the non-cleaved form and the non-cleaved control.

[0111] Figure 79 The results of TDCC assays using a dual-targeting construct against EGFR and EpCAM (sometimes referred to herein as "iso-COBRA") employing the EGFR2 binding domain and the hVIB664 EpCAM binding domain are depicted. The results show that MMP9-cleaved Pro623 is more effective than uncleaved Pro623 or the non-cleavable control Pro625.

[0112] Figure 80 The results of TDCC assays using a dual-targeting construct targeting EGFR and EpCAM with the EGFR2-binding domain and the hVIB665 EpCAM-binding domain are depicted. The results show that MMP9-cleaved Pro624 is more effective than cleaved Pro624 or the non-cleavable control Pro626.

[0113] Figure 81 TDCC assays were performed using a dual-targeting construct (oriented opposite to Pro624) employing the hEGFR2 binding domain and the hVIB665 EpCAM binding domain. Results showed that MMP9-cleaved Pro698 was more effective than cleaved Pro698 or the non-cleavable control Pro699.

[0114] Figure 82 The results of TDCC assays for a dual-targeting construct using the hF7 B7H3 binding domain and the hVIB664 EpCAM binding domain in Pro655 are depicted. The results show that Pro665 with MMP9 cleavage is more effective than either uncleaved Pro665 or the non-cleavable control Pro659.

[0115] Figure 83 The results of TDCC assays using a dual-targeting construct targeting both B7H3 and EpCAM in Pro657 (with the opposite orientation to Pro655) are depicted. The results show that MMP9-cleaved Pro657 is more effective than cleaved Pro657 or the non-cleavable control Pro661.

[0116] Figure 84The results of TDCC assays for a dual-targeting construct using the hF7 B7H3 binding domain and the hVIB665 EpCAM binding domain in Pro656 are depicted. The results show that MMP9-cleaved Pro656 is more effective than cleaved Pro656 or the non-cleavable control Pro660.

[0117] Figure 85 The results of TDCC assays using a dual-targeting construct targeting both B7H3 and EpCAM in Pro658 (with the opposite orientation to Pro656) are depicted. The results show that MMP9-cleaved Pro658 is more effective than cleaved Pro658 or the non-cleavable control Pro662.

[0118] Figure 86 The described experiments showed that the dual-targeting constructs Pro656 and Pro658, which bind to both B7H3 and EpCAM (with different orientations of the two domains), killed all three cell types (those expressing B7H3 but not EpCAM, those expressing EpCAM but not B7H3, and those expressing both). In contrast, Pro225 (which has two anti-B7H3 domains) killed only two cell types (those expressing both B7H3 and EpCAM). Similarly, Pro566 (which has two anti-EpCAM domains) killed only two cell types: those expressing both EpCAM and B7H3 and EpCAM. The Raji F cell line transiently expressing the appropriate proteins was used.

[0119] Figure 87 The results of TDCC assays using the B7H3-targeted construct Pro226 are depicted. The results show that Pro226 and Pro226 cleaved with MMP9 exhibited higher activity compared to the non-cleavable control Pro296. Pre-cleaved Pro226 showed an EC50 of approximately 8 pM.

[0120] Figure 88 The results of a TDCC assay using Pro226, expressed in the presence of tunicamycin, are depicted, which enhances the potency of Pro226. Taninomycin inhibits glycosylation, and the EC50 of the cleavage products increases to 1 pM, indicating that glycosylation is decreasing EC50 (increasing potency).

[0121] Figure 89The results of TDCC assays using Pro664 are depicted. Compared to Pro226 (the same construct but without an amino acid variant), Pro664 contains an anti-B7H3 hF12 domain with an amino acid variant (S59Y) to remove glycosylation sites. Pro664 exhibits similar activity to Pro226.

[0122] Figure 90 The results of TDCC assays using Pro665 are depicted. Compared to Pro226 (the same construct but without an amino acid variant), Pro665 contains an anti-B7H3 hF12 domain with an amino acid variant (N57Q) to remove glycosylation sites. Pro665 shows higher potency than Pro226.

[0123] Figure 91 The results of TDCC assays using Pro667 are depicted. Compared to Pro226 (the same construct but without an amino acid variant), Pro667 contains an anti-B7H3 hF12 domain with an amino acid variant (N57E) to remove glycosylation sites. Pro667 showed lower potency than Pro226.

[0124] Figure 92 The results of TDCC assays using Pro694 are depicted. Compared to Pro226 (the same construct but without an amino acid variant), Pro694 contains an anti-B7H3 hF12 domain with an amino acid variant (S59A) to remove glycosylation sites. Pro694 shows higher potency than Pro226.

[0125] Figure 93 The results of TDCC assays using Pro695 are depicted. Compared to Pro226 (the same construct but without an amino acid variant), Pro695 contains an anti-B7H3 hF12 domain with an amino acid variant (N57D) to remove glycosylation sites. Pro695 exhibits similar potency to Pro226.

[0126] Figure 94 The TDCC assay is shown, comparing two different inactivations in the inactive domains. Pro186 showed label inactivation in both Vhi and Vli, and Pro476 showed i2 inactivation, and they exhibited similar potency.

[0127] Figure 95 The TDCC assays of Pro186 containing the MMP9 cleavage site and Pro393 containing the S9 cleavage site are shown; the results indicate that the two constructs behave similarly at different protease cleavage sites.

[0128] Figure 96The TDCC assays of Pro186 containing the MMP9 cleavage site and Pro394 containing the ST14 MV cleavage site were compared; the results showed that the two constructs behaved similarly at different protease cleavage sites.

[0129] Figure 97 The TDCC assays of Pro186 containing the MMP9 cleavage site and Pro395 containing the CathS cleavage site were compared; the results showed that the two constructs behaved similarly at different protease cleavage sites.

[0130] Figure 98 TDCC assays were performed comparing Pro186 containing the MMP9 cleavage site and Pro396 containing the MMP9v cleavage site; the results showed that the two constructs behaved similarly with different protease linker sequences. Note that the MMP9 site is cleaved by both MMP9 and CathS, while MMP9v is MMP9-specific.

[0131] Figure 99 The TDCC assays of Pro186 containing the MMP9 cleavage site and Pro429 containing the MepGzb cleavage site were compared; the results showed that the two constructs behaved similarly at different protease cleavage sites.

[0132] Figure 100 The TDCC assays of Pro186 containing the MMP9 cleavage site and Pro430 containing the MMP9-2 cleavage site were compared; despite the different MMP9 cleavage sites, their performance was similar.

[0133] Figure 101 The TDCC assays of Pro186 containing the MMP9 cleavage site and Pro431 containing the ST14MS cleavage site were compared; the results showed that the two constructs behaved similarly at different protease cleavage sites.

[0134] Figure 102 TDCC assays using Pro676 (a Trop2-containing construct) showed that Pro677 and its active dimer (Pro684AD) exhibited higher activity against BXPC3 tumor cells compared to Pro680 (an uncleavable control). The active dimer was prepared by expressing a self-dimerizing active domain.

[0135] Figure 103 The TDCC assay using Pro677 (a construct containing Trop2) showed that Pro677 and its active dimer (Pro685AD) exhibited higher activity against BXPC3 tumor cells compared to Pro681 (an uncleavable control).

[0136] Figure 104 The TDCC assay using Pro677 (a construct containing Trop2) showed that Pro677 and its active dimer (Pro685AD) exhibited higher activity against HT29 tumor cells compared to Pro681 (a non-lytic control). Pro677 showed less activation by HT29 cells compared to BXPC3 cells.

[0137] Figure 105 The TDCC assay using Pro678 (a construct containing Trop2) showed that Pro678 and its active dimer (Pro686AD) exhibited higher activity against BXPC3 tumor cells compared to Pro682 (a non-cleavable control).

[0138] Figure 106 The TDCC assay using Pro679 (a construct containing Trop2) showed that Pro679 and its active dimer (Pro687 active domain, AD) exhibited higher activity against BXPC3 tumor cells compared to Pro683 (an uncleavable control).

[0139] Figure 107 The TDCC assay using Pro808 (a construct containing Trop2) showed that Pro808, its cleaved form, and the active dimer (Pro810AD) exhibited higher activity against BXPC3 tumor cells compared to Pro809 (an uncleaved control).

[0140] Figure 108 The TDCC assay using Pro808 (a Trop2-containing construct) was depicted in HT29 tumor cells. Pro810AD (the active domain) showed higher activity in the TDCC assay than Pro809 (a non-cleavable control). Full-length Pro808 did not show much activation in the assay using HT29 cells.

[0141] Figure 109 The TDCC assay using Pro819 (a construct containing Trop2) showed that Pro819 and its active dimer (Pro821AD) exhibited higher activity against BXPC3 tumor cells compared to Pro820 (an uncleavable control).

[0142] Figure 110TDCC assays using Pro819 (a construct containing Trop2) showed that the active dimer (Pro821AD) exhibited higher activity compared to the non-cleavable control. Full-length Pro819 did not show significantly more HT29 cell activation compared to the non-cleavable control Pro820. Figure 107 and 108 as well as Figure 109 and 110 The two pairs of data indicate that, when combined with data showing that adding an MMP9 inhibitor reduces activity, different target cell types produce different levels of MMP activity (see [reference]). Figure 133 ).

[0143] Figure 111 The TDCC assay using Pro311 (a FOLR1 construct) showed that Pro311 cleaved by MMP9 exhibited higher activity against H292 tumor cells compared to the non-cleavable control Pro299.

[0144] Figure 112 The TDCC assay using Pro312 (a FOLR1 construct) showed that Pro312 cleaved by MMP9 exhibited higher activity against H292 tumor cells compared to the non-cleavable control Pro303.

[0145] Figure 113 Results of TDCC assays using the dual-targeting construct Pro420 with FOLR1 and EGFR targeting are depicted. The results show that Pro420 and Pro420 cleaved with MMP9 exhibit higher activity compared to the non-cleavable control Pro299.

[0146] Figure 114 Results of TDCC assays using the dual-targeting construct Pro421, which has FOLR1 and EGFR targets (in the opposite orientation to Pro420), are depicted. The results show that Pro421 and Pro421 cleaved with MMP9 exhibit higher activity compared to the cleavable control Pro299.

[0147] Figure 115 The results of TDCC assays using the dual-targeting construct Pro551 with EGFR and FOLR1 targeting are depicted. The results show that Pro551 and Pro551 cleaved with MMP9 exhibit higher activity compared to the non-cleavable control Pro550.

[0148] Figure 116Results of TDCC assays using the dual-targeting construct Pro552, which has FOLR1 and EGFR targets (in the opposite orientation to Pro551), are depicted. The results show that Pro522 and Pro522 cleaved with MMP9 exhibit higher activity compared to the cleavable control Pro303.

[0149] Figure 117 The results of a TDCC assay using the dual-targeting construct Pro254 are depicted, where each targeting domain binds to a different epitope of EGFR and is effective against HT29 cells expressing EGFR.

[0150] Figure 118 The results of TDCC assays using two different dual-targeting constructs, which also use two different targeting domains but both bind to B7H3, are depicted. Pro479 and Pro480 are identical except for the orientation of the two binding domains, and both showed activity upon MMP9 lysis in B7H3-expressing HT29 cells.

[0151] Figure 119 The results of TDCC assays using Pro233 lysed on EGFR-expressing HT-29 cells are depicted. Pro233 showed good conditionability and good power during lysis with MMP9.

[0152] Figure 120 This study describes the results of a tumor regression study using Pro225 (a construct with B7H3 targeting and an MMP9 linker). The study was conducted using a human PBMC implantation model, in which human PBMCs were intravenously implanted into NSG-β2M- / - mice (Jackson); three days post-implantation, tumor cell lines were subcutaneously implanted into the mice. Once tumor growth was established, mice were randomly assigned based on tumor volume and administered the test substance intravenously as shown. Tumor volume was assessed by caliper measurement. The results showed that Pro225 induced regression of established solid tumors compared to the non-lytic control Pro295.

[0153] Figure 121 The results of a tumor regression study using Pro226 (a construct with B7H3 targeting and an MMP9 linker) are described. The study is as follows... Figure 120 The procedure was performed as described above, and the results showed that Pro226 caused established solid tumors to regress compared to the non-lytic control Pro295.

[0154] Figure 122The results of a tumor regression study using Pro565 and Pro566, both of which have EpCAM targeting and an MMP9 linker, are described. The results showed that both Pro565 and Pro566 exhibited an antitumor response compared to the non-cleavable control Pro568.

[0155] Figure 123 Depicting the results of tumor regression studies using Pro393, which utilizes EGFR targeting and the S9 linker, Pro393... Figure 122 The protocol described in [the document] was followed. Results showed that Pro393 caused regression of established solid tumors compared to the non-lytic control Pro214.

[0156] Figure 124 Depicting the results of a tumor regression study using Pro394, which utilizes EGFR targeting and the ST14 MV linker, using Figure 122 The protocol described in [the document] was followed. Results showed that Pro394 exhibited a smaller antitumor response compared to the non-cleavable control Pro214.

[0157] Figure 125 Depicting the results of a tumor regression study using Pro395, which utilizes EGFR targeting and the CathS linker, using Figure 122 The protocol described in [the document] was followed. Results showed that Pro395 caused regression of established solid tumors compared to the non-lytic control Pro214.

[0158] Figure 126 Depicting the results of a tumor regression study using Pro396, which utilizes EGFR targeting and the MMP9v linker, using Figure 122 The protocol described in [the document] was followed. Results showed that Pro396 exhibited an antitumor response compared to the non-cleavable control Pro214.

[0159] Figure 127 Depicting the results of a tumor regression study using Pro430, which utilizes EGFR targeting and the MMP9-2 linker, using Figure 122 The protocol described in [the document] was followed. Results showed that Pro430 caused regression of established solid tumors compared to the non-lytic control Pro214.

[0160] Figure 128 Depicting the results of a tumor regression study using Pro431, which utilizes EGFR targeting and the ST14 MS linker, using Figure 122 The protocol described in [the document] was followed. Results showed that Pro431 exhibited an antitumor response compared to the non-cleavable control Pro214.

[0161] Figure 129 Depicting the results of tumor regression studies using Pro476, which utilizes EGFR targeting, the MMP9-2 linker, and the inactive domains Vli2 and VHi2, Pro476... Figure 122 The protocol described in [the document] was followed. Results showed that Pro476 caused regression of established solid tumors compared to the non-lytic control Pro214.

[0162] Figure 130 Depicting the results of a tumor regression study using Pro517, which utilizes EGFR targeting and the MMP9-2 linker, using... Figure 122 The protocol described in [the document] was followed. Results showed that Pro517 caused regression of established solid tumors compared to the non-lytic control Pro513.

[0163] Figure 131 Depicting the results of a tumor regression study using Pro664, which utilizes B7H3 targeting and the MMP9 linker, using Figure 120 The protocol described in [the document] was followed. Results showed that Pro664 caused regression of established solid tumors compared to the non-lytic control Pro766.

[0164] Figure 132 Depicting the results of a tumor regression study using Pro676, which utilizes Trop2 targeting and the MMP9 linker, Pro676... Figure 122 The protocol described in [the document] was followed. Results showed that Pro676 caused regression of established solid tumors compared to the non-lytic control Pro681.

[0165] Figure 133 The results of studies using Pro225 (an anti-B7H3 construct with an MMP9 linker) and increased amounts of the MMP-specific inhibitor batimastat showed that the potency of unlysed Pro225 was reduced by batimastat, thus demonstrating that COBRA was lysed and activated in the assay. Detailed Implementation

[0166] introduction

[0167] This invention relates to methods for reducing the toxicity and side effects of bispecific antibodies (including antibody-like functional proteins) that bind to important physiological targets such as CD3 and tumor antigens. Many antigen-binding proteins (such as antibodies) can have significant side effects due to targeting normal tissues, and therefore it is necessary to activate the binding ability of therapeutic molecules only near diseased tissues to avoid interactions with normal tissues. Therefore, this invention relates to multivalent conditionally effective (“MCE”) proteins having a number of functional protein domains. Generally, one of these domains is an antigen-binding domain (ABD) that binds to a target tumor antigen (TTA), and another is an ABD that binds to T-cell antigens such as CD3 under certain conditions. Additionally, MCE proteins also include one or more protease cleavage sites. That is, therapeutic molecules are prepared in a “prodrug”-like form, wherein the CD3-binding domain is inactive before exposure to the tumor environment. The tumor environment contains proteases, such that upon exposure to the proteases, the prodrug is cleaved and becomes active.

[0168] This is typically achieved by using proteins containing “pseudo” variable heavy-restructuring domains and “pseudo” variable light-restructuring domains targeting T-cell antigens such as CD3, which confine the CD3Fv of the MCE to an inactive form as discussed herein. Because TTA targets the MCE near the tumor, it exposes the MCE to the protease. Upon cleavage, the active variable heavy-restructuring domain and active light-restructuring domain are now able to pair to form one or more active ABDs targeting CD3, and thus recruit T cells to the tumor for treatment.

[0169] Generally, CD3-binding domains (“Fv”) are in a constrained form, where the linker between the active variable heavy domain and the active variable light domain that traditionally form the Fv is too short to allow the two active variable domains to bind to each other; this is called a “constrained linker”; these can be constrained and cleavable (CCL, as used in Form 1) or constrained and non-cleavable (CNCL, as used in Form 2). Instead, in the form of prodrugs (e.g., uncleaved), prodrug peptides also contain a “pseudo-Fv domain.” The pseudo-Fv domain contains variable heavy and light chain domains with standard frame regions but “inert” or “non-active” CDRs. The pseudo-Fv domain also has a constrained linker between the inactive variable heavy chain domain and the inactive variable light chain domain. Because neither the Fv nor the pseudo-Fv domain can self-assemble due to spatial constraints, intramolecular assembly exists to pair aVL with iVH and aVH with iVL due to the affinity of their respective frame regions. However, due to the "inert" CDR of the pseudo-domain, the resulting ABD will not bind CD3, thus preventing toxicity outside of diseased tissue (such as tumors). However, in the presence of proteases in or near the tumor, the prodrug construct is cleaved, releasing the pseudo-Fv domain from the surface, and thus allowing intermolecular association between the "real" variable heavy and variable light domains (e.g., two cleaved constructs binding together), thereby triggering active CD3 binding and the resulting tumor efficacy. These constructs are generally referred to herein as conditionally bispecific retargeting activation constructs or "COBRA". TM The stability of intramolecular assembly was demonstrated by conditional experiments in this paper, showing that in the absence of proteases, the uncleaved constructs are inactive (e.g., do not form an active CD3-binding domain).

[0170] Interestingly, for ease of description, although these constructs are all referred to as "constrained" herein, other work shows that even if one of the Fv domains is unconstrained, for example, one of the domains may have a longer, more flexible linker, which is beneficial for intramolecular assembly. That is, as Figures 37-39 As shown, if only one Fv domain is constrained (a domain with active VL and VH or a pseudo-Fv domain), intramolecular assembly still occurs (e.g., an uncleaved construct is inactive without protease cleavage). However, in the current system, the protein is better expressed when both linkers are constrained. However, as those skilled in the art will understand, any form 1, form 2, or form 4 construct herein may have one of these Fv domains with “unconstrained” or “flexible” linkers. For ease of reference, a construct having two Fv domains in a constrained form is shown.

[0171] The construction and form of this invention are variations of the invention described in WO2017 / 156178, which is hereby expressly incorporated herein by reference in its entirety. Figure 17-21 As shown, the previous construct possessed isomerization capabilities due to the presence of two sets of VH and VL domains within a single polypeptide, resulting in both a bivalent scFv and a single-chain biantibody. Even after purification of each isoform, the bivalent construct remained in equilibrium with the biantibody isoform. The utility of the construct was reduced because the single-chain biantibody had the ability to bind to CD3 in the absence of protease cleavage.

[0172] To address this problem, the present invention provides four separate types of constructs to accomplish this conditional activation. Prodrug activation can occur in one of four general ways, as generally illustrated in the accompanying drawings. Figure 1 The “Form 1” mechanism is illustrated in this embodiment. In this embodiment, the prodrug construct has two cleavage sites: one between the VH and vl domains of the constrained Fv, thereby releasing two variable domains for association; and a second at the site where a pseudo-Fv domain is released from the prodrug construct, leaving two molecules associated due to the innate self-assembly of the variable heavy and variable light domains, each of which also has an antigen-binding domain for tumor antigens, thereby allowing T cells to be recruited to the tumor site.

[0173] In the alternative implementation scheme, the prodrug construct is shown as follows: Figure 2 In this embodiment, the domain linker between the active variable heavy chain and the active light chain is a constrained but non-cleavable linker (“CNCL”). In the prodrug form, the inactive VH and VL of the constrained pseudo-Fv domain associate with the VH and VL of the constrained Fv domain, resulting in no CD3 binding. However, once cleavage occurs in the tumor setting, the two distinct activated proteins (each containing an active variable heavy chain and a light chain domain) associate to form two anti-CD3 binding domains. This form 2 has two target tumor antigen-binding domains (“TTA-ABD”), as described in more detail below, which may be identical (e.g., “iso-COBRA”) or different (e.g., “iso-COBRA”). If different, they may each target different tumor antigens, or they may target the same tumor antigen but different epitopes, as described more fully below.

[0174] In addition to the "single-chain protein" COBRA form discussed above (where all components are contained in a single amino acid sequence), there are also "half-COBRA" constructs that depend on pairwise action, as shown in Figure 3. In this embodiment, each protein has an active variable domain and an inert variable domain separated by a protease cleavage site. Each molecule contains a TTA-binding domain such that when the molecule binds to TTA and is exposed to tumor proteases, the inert domain is cleaved, and the two active variable domains self-assemble to form an anti-CD3 binding domain.

[0175] Furthermore, this invention also provides a "Form 4" construct, such as Figure 4 As described in the text. These are similar to the “Form 2” design, except that they use a single ABD targeting TTA, so that upon cleavage, the two prodrug molecules now form a tetravalent bispecific construct containing two active anti-CD3 domains, as further described below.

[0176] Therefore, the forms and constructions of the present invention can be used to treat diseases.

[0177] definition

[0178] To provide a more complete understanding of this application, several definitions are presented below. These definitions are intended to cover syntactic equivalents.

[0179] As used herein, “amino acid” and “amino acid identity” mean one of the 20 naturally occurring amino acids or any non-natural analogue that may be present at a specific, defined location. In many embodiments, “amino acid” refers to one of the 20 naturally occurring amino acids. As used herein, “protein” means at least two covalently attached amino acids, including proteins, polypeptides, oligopeptides, and peptides.

[0180] As used herein, “amino acid modification” means an alteration of a portion of a polypeptide sequence by substitution, insertion, and / or deletion of amino acids or by chemical attachment to a protein. For example, a modification can be an altered carbohydrate or PEG structure attached to a protein. For clarity, unless otherwise noted, amino acid modifications always refer to amino acids encoded by DNA, such as the 20 amino acids that form a codon in DNA and RNA. Preferred amino acid modifications in this document are substitutions.

[0181] As used herein, “amino acid substitution” or “replacement” means that an amino acid at a specific position in the parental polypeptide sequence is replaced by a different amino acid. In particular, in some embodiments, the substitution is an amino acid that is not naturally present at the specific position, or is not naturally present in an organism or in any organism. For clarity, a protein that has been engineered to alter the nucleic acid coding sequence but not the starting amino acid (e.g., exchanging CGG (encoding arginine) for CGA (still encoding arginine) to increase expression levels in a host organism) is not an “amino acid substitute”; that is, although a new gene encoding the same protein is generated, if the protein has the same amino acid at the specific position where it begins, the protein is not an amino acid substitute.

[0182] As used in this article, "amino acid insertion" or "insertion" means the addition of an amino acid sequence at a specific position in the parental polypeptide sequence.

[0183] As used in this article, "amino acid deletion" or "deletion" means the removal of an amino acid sequence at a specific position in the parental polypeptide sequence.

[0184] As summarized herein, the peptides of the present invention specifically bind to CD3 and target tumor antigens (TTAs), such as target cell receptors. "Specific binding," "specifically bound to," or "specific to a particular antigen or epitope" refers to binding that is distinctly different from non-specific interactions. Specific binding can be measured, for example, by measuring molecular binding compared to binding with a control molecule, which is typically a similarly structured molecule that does not have binding activity. For example, specific binding can be measured by competition with a control molecule similar to the target.

[0185] Specific binding to a particular antigen or epitope can, for example, be achieved by a KD of at least about 10 for the antigen or antigenic epitope. -4 M, at least about 10 -5 M, at least about 10 -6 M, at least about 10 -7 M, at least about 10 -8 M, at least about 10 -9 M, or at least about 10 -10 M, at least about 10 -11 M, at least about 10 -12 M or higher antibody expression, where KD refers to the dissociation rate of a specific antibody-antigen interaction. Typically, the KD of an antibody that specifically binds to an antigen is 20, 50, 100, 500, 1000, 5,000, 10,000 or higher relative to the antigen or epitope.

[0186] Furthermore, specific binding to a particular antigen or epitope can be demonstrated, for example, by antibodies whose KA or Ka for the antigen or epitope is at least 20, 50, 100, 500, 1000, 5,000, 10,000, or higher folds than that for a control, where KA or Ka refers to the association rate of the specific antibody-antigen interaction. Binding affinity is typically measured using the Biacore assay or Octet known in the art.

[0187] As used herein, “parental polypeptide” or “precursor polypeptide” (including Fc parents or precursors) means a polypeptide that is subsequently modified to produce a variant. The parental polypeptide can be a naturally occurring polypeptide, or a variant or engineered form of a naturally occurring polypeptide. A parental polypeptide can refer to the polypeptide itself, a composition comprising the parental polypeptide, or the amino acid sequence encoding the polypeptide. Therefore, as used herein, “parental Fc polypeptide” means an unmodified Fc polypeptide that has been modified to produce a variant, and as used herein, “parental antibody” means an unmodified antibody that has been modified to produce a variant antibody.

[0188] As used in this article, "position" refers to a location within the sequence of a protein. Positions can be numbered sequentially or according to established formats, such as the EU index used for antibody numbering.

[0189] As used herein, “target antigen” means a molecule that is specifically bound to the variable region of a given antibody. Target antigens can be proteins, carbohydrates, lipids, or other chemical compounds. A range of suitable exemplary target antigens are described herein.

[0190] As used herein, “target cell” refers to a cell that expresses the target antigen. Generally, for the purposes of this invention, target cells are tumor cells that express TTA or T cells that express CD3 antigen.

[0191] As used herein, the biological term "Fv," "Fv sheet domain," or "Fv region" refers to a polypeptide containing VL and VH domains, typically derived from an antigen-binding domain of an antibody. If the Fv domain contains active VH and VL domains, it typically forms an "antigen-binding domain" or "ABD" as discussed herein (however, in some cases, Fv domains with constrained linkers are used so that an active ABD is not formed prior to cleavage). As discussed below, in this invention, the Fv domain can be configured in various ways and can be "active" or "inactive," such as in scFv form, constrained Fv form, pseudo-Fv form, etc. It should be understood that in this invention, in some cases, the Fv domain consists of VH and VL domains on a single polypeptide chain, such as... Figure 1 and Figure 2As shown, but with a constrained junction, intramolecular ABDs cannot be formed. In these embodiments, two active ABDs are formed after cleavage. In some cases, the Fv domain consists of VH and VL domains, one of which is inert, so that intermolecular ABDs are formed only after cleavage. As discussed below, in this invention, the Fv domain can be configured in various ways and can be “active” or “inactive,” such as in the form of scFv, constrained Fv, pseudo-Fv, etc. Furthermore, as discussed herein, Fv domains containing VH and VL can be / form ABDs, and other ABDs not containing VH and VL domains can be formed using sdABDs.

[0192] The term "variable region" in this document refers to a region of an immunoglobulin containing one or more Ig domains, which are essentially encoded by any Vκ, Vλ, and / or VH genes that constitute the κ, λ, and heavy and light chain immunoglobulin loci, respectively. In some cases, a single variable domain may be used, such as sdFv (also referred to herein as sdABD).

[0193] In embodiments utilizing both variable heavy (VH) and variable light (VL) domains, each VH and VL consists of three hypervariable regions (“complementarity-determining regions”, “CDRs”) and four “framework regions” or “FRs”, arranged in the following order from the amino terminus to the carboxyl terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Thus, the VH domain has the structure vhFR1-vhCDR1-vhFR2-vhCDR2-vhFR3-vhCDR3-vhFR4, and the VL domain has the structure vlFR1-vlCDR1-vlFR2-vlCDR2-vlFR3-vlCDR3-vlFR4. As described more fully herein, the vhFR and vlFR regions self-assemble to form the Fv domain. Generally, in the prodrug form of the present invention, there exists a “constrained Fv domain” in which the VH and VL domains cannot self-associate, and a “pseudo-Fv domain” in which the CDR does not form an antigen-binding domain when self-associating.

[0194] The hypervariable region confers antigen-binding specificity and typically comprises approximately amino acid residues 24-34 (LCDR1; "L" indicates light chain), 50-56 (LCDR2), and 89-97 (LCDR3) from the light chain variable region and approximately 31-35B (HCDR1; "H" indicates heavy chain), 50-65 (HCDR2), and 95-102 (HCDR3) from the heavy chain variable region; Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, Public Health Service, 5th edition, National Institutes of Health, Bethesda Md. (1991) and / or those residues forming the hypervariable ring in the light chain variable region (e.g., residues 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3)) and those residues forming the hypervariable ring in the heavy chain variable region, 26-32 (HCDR1), 53-55 (HCDR2), and 96-101 (HCDR3); Chothia and Lesk (1987) J. Mol. Biol. 196: 901-917. Specific CDRs of the present invention are described below.

[0195] As those skilled in the art will understand, the exact numbering and location of CDRs may differ in different numbering systems. However, it should be understood that the disclosure of variable-weight and / or variable-light sequences includes the disclosure of the associated (inherent) CDR. Therefore, the disclosure of each variable-weight region is the disclosure of vhCDRs (e.g., vhCDR1, vhCDR2, and vhCDR3), and the disclosure of each variable-light region is the disclosure of vlCDRs (e.g., vlCDR1, vlCDR2, and vlCDR3).

[0196] A useful comparison of CDR numbers is as follows, see Lafranc et al., Dev. Comp. Immunol. 27(1): 55-77 (2003):

[0197] Table 1

[0198] Kabat+Chothia IMGT Kabat AbM Chothia Contact vhCDR1 26-35 27-38 31-35 26-35 26-32 30-35 vhCDR2 50-65 56-65 50-65 50-58 52-56 47-58 vhCDR3 95-102 105-117 95-102 95-102 95-102 93-101 vlCDR1 24-34 27-38 24-34 24-34 24-34 30-36 vlCDR2 50-56 56-65 50-56 50-56 50-56 46-55 vlCDR3 89-97 105-117 89-97 89-97 89-97 89-96

[0199] Throughout this specification, when referring to residues in the variable domains (approximately residues 1-107 of the light chain variable region and residues 1-113 of the heavy chain variable region), the Kabat numbering system and the EU numbering system of the Fc region are generally used (e.g., Kabat et al., ibid. (1991)).

[0200] This invention provides a large number of different sets of CDRs. In this case, in the context of the anti-CD3 component, the "full CDR set" comprises three variable light CDRs and three variable heavy CDRs, such as vlCDR1, vlCDR2, vlCDR3, vhCDR1, vhCDR2, and vhCDR3. As those skilled in the art will understand, each set of CDRs, namely VH and VL CDRs, can bind to the antigen individually or as a group. For example, in the constrained Fv domain, vhCDRs can bind to CD3, and vlCDRs can bind to CD3, but in the constrained form, they cannot bind to CD3.

[0201] In the context of a single-domain ABD (“sdABD”), as commonly used in this paper for binding to a target tumor antigen (TTA), the CDR group consists of only three CDRs; these are sometimes referred to in the art as “VHH” domains.

[0202] These CDRs can be part of a larger variable light domain or a variable heavy domain. Furthermore, as outlined more comprehensively in this paper, in the case of scFv sequences, the variable heavy domain and variable light domain can be on separate polypeptide chains or on a single polypeptide chain, depending on the form and configuration described in this paper.

[0203] CDR promotes the formation of antigen-binding sites, or more particularly epitope-binding sites. An "epitaph" is a determinant that interacts with a specific antigen-binding site called a paratope in a variable region. Antigenic epitopes are groups of molecules, such as amino acid or sugar side chains, and typically possess specific structural and charge properties. A single antigen may have more than one epitope.

[0204] Epitopes can contain amino acid residues that directly participate in binding (also known as the immunodominant component of the epitope) as well as other amino acid residues that do not directly participate in binding, such as amino acid residues that can be effectively blocked by a specific antigen-binding peptide; in other words, the amino acid residues are within the footprint of the specific antigen-binding peptide.

[0205] Epitopes can be conformational or linear. Conformational epitopes are generated by spatially aligned amino acids in different segments of a linear polypeptide chain. Linear epitopes are generated by adjacent amino acid residues in a polypeptide chain. The possible difference between conformational and non-conformational antigenic epitopes is that, in the presence of denaturing solvents, they lose binding to the former but retain binding to the latter.

[0206] Epitopes typically comprise at least three, and more often at least five, or eight to ten amino acids in a unique spatial conformation. Antibodies that recognize the same epitope can be validated by a simple immunoassay demonstrating the ability of one antibody to block the binding of another antibody to a target antigen, such as "binning." As outlined below, the invention includes not only the antigen-binding domains and antibodies listed herein, but also those that compete for binding with epitopes bound by the listed antigen-binding domains.

[0207] The variable heavy and variable light structural domains of the present invention can be "active" or "inactive".

[0208] As used herein, “inactive VH” (“iVH”) and “inactive VL” (“iVL”) refer to components of a pseudo-Fv domain that, when paired with their respective homologous VL or VH partners, form VH / VL pairs that nonspecifically bind to the antigen to which an “active” VH or “active” VL would bind if it were to bind to a non-“inactive” similar VL or VH. Exemplary “inactive VH” and “inactive VL” domains are formed by mutations in the wild-type VH or VL sequence, as outlined more fully below. Exemplary mutations are located within CDR1, CDR2, or CDR3 of the VH or VL. Exemplary mutations include placing a domain linker within CDR2, thereby forming an “inactive VH” or “inactive VL” domain. In contrast, an "active VG" or "active VL" is one that can specifically bind to its target antigen when paired with its "active" homologous partner, namely VL or VH, respectively. Therefore, it should be understood that a pseudo-Fv can be a VH / iVL pair, an iVH / VL pair, or an iVH / iVL pair.

[0209] In contrast, as used herein, the term "active" refers to a CD-3 binding domain capable of specifically binding to CD-3. This term is used in two cases: (a) when referring to a single member of an Fv binding pair (i.e., VH or VL) having a sequence capable of pairing with its homologous partner and specifically binding to CD-3; and (b) a pair of homologs (i.e., VH and VL) capable of specifically binding to CD-3. Exemplary "active" VH, VL, or VH / VL pairs are wild-type or parental sequences.

[0210] "CD-x" refers to a group of differentiation (CD) proteins. In an exemplary embodiment, CD-x is selected from those CD proteins that have an effect on the recruitment or activation of T cells in a subject who has been administered the polypeptide construct of the present invention. In one exemplary embodiment, CD-x is CD3, the sequence of which is shown in Figure 5.

[0211] The term "binding domain" in this invention characterizes a domain that (specifically) binds to / interacts with / recognizes a given target epitope or site on a target molecule (antigen) (e.g., EGFR and CD-3, respectively). The structure and function of the target antigen-binding domain (recognizing EGFR), and preferably the same for the CD-3-binding domain (recognizing CD3), are based on the structure and / or function of the antibody (e.g., full-length or whole immunoglobulin molecule, including sdABD). According to the invention, the target antigen-binding domain is generally characterized by the presence of three CDRs binding to the target tumor antigen (commonly referred to in the art as variable heavy domains, although corresponding light chain CDRs are not present). Alternatively, the ABD for TTA may contain three light chain CDRs (i.e., CDR1, CDR2, and CDR3 in the VL region) and / or three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3 in the VH region). The CD-3-binding domain preferably also contains at least the minimum structural requirements of an antibody that allows target binding. More preferably, the CD-3 binding domain comprises at least three light chain CDRs (i.e., CDR1, CDR2, and CDR3 in the VL region) and / or three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3 in the VH region). It is envisioned that in exemplary embodiments, the target antigen and / or the CD-3 binding domain are generated or obtained through phage display or library screening methods.

[0212] As used herein, "domain" refers to a functional protein sequence, as outlined herein. The domains of this invention include a tumor target antigen binding domain (TTA domain), a variable heavy domain, a variable light domain, an scFv domain, a linker domain, and a half-life extension domain.

[0213] In this article, "domain linker" refers to an amino acid sequence that connects two domains as outlined herein. Domain linkers can be cleavable linkers, constrained cleavable linkers, non-cleavable linkers, constrained non-cleavable linkers, scFv linkers, etc.

[0214] As used herein, a “cleavable linker” (“CL”) refers to an amino acid sequence that can be cleaved by proteases, preferably human proteases, in diseased tissues as outlined herein. Cleavable linkers are typically at least 3 amino acids in length, with 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acids permitted for use in this invention, depending on the desired flexibility. Many cleavable linker sequences are found in Figures 6 and 5.

[0215] In this article, "non-cleavable linker" ("NCL") refers to an amino acid sequence that cannot be cleaved by human proteases under normal physiological conditions.

[0216] In this document, a “constrained cleavable linker” (“CCL”) refers to a short polypeptide containing a protease cleavage site (as defined herein) connecting two domains as outlined herein, such that the two domains cannot significantly interact with each other until they reside on different polypeptide chains, for example, in a cleaved manner. When a CCL connects VH and VL domains as defined herein, VH and VL cannot self-assemble to form a functional Fv before cleavage due to spatial constraints in an intramolecular manner (however, they can assemble intermolecularly into a pseudo-Fv domain). After cleavage by the relevant protease, VH and VL can assemble intermolecularly to form an active antigen-binding domain. Generally, the length of a CCL is less than 10 amino acids, of which 9, 8, 7, 6, 5, and 4 amino acids are used in this invention. Generally, the length of the protease cleavage site is typically at least 4+ amino acids to impart sufficient specificity, as shown in Figure 6.

[0217] In this article, "constrained non-cleavable linker" ("CNCL") refers to a short polypeptide in which two domains, as outlined herein, are linked in a manner in which they cannot significantly interact with each other and are not significantly cleaved by human proteases under physiological conditions.

[0218] In this document, "constrained Fv domains" refers to Fv domains comprising an active variable heavy domain and an active variable light domain, wherein the Fv domains are covalently linked to a constrained linker as outlined herein in such a manner that the active heavy and light variable domains cannot interact intramolecularly to form an active Fv that binds an antigen (such as CD3). Therefore, constrained Fv domains are similar to scFv domains but, due to the presence of the constrained linker, cannot bind antigens (although they can intermolecularly assemble with inert variable domains to form pseudo-Fv domains).

[0219] In this article, "pseudo-Fv domain" refers to a domain containing a pseudo or inactive variable heavy domain or a pseudo or inactive variable light domain linked by domain linkers (which can be cleavable, constrained, non-cleavable, unconstrained, etc.). The iVH and iVL domains of the pseudo-Fv domain do not bind to human antigens when associated with each other (iVH / iVL) or when associated with active VH or VL; therefore, the iVH / iVL, iVH / VL, and iVL / VH Fv domains do not readily bind to human proteins, making these domains inert in the human body.

[0220] In this paper, "single-chain Fv" or "scFv" refers to a variable-weight (VH) domain that is typically covalently attached to a variable-light (VL) domain using an scFv joint as discussed herein to form an scFv or scFv domain. The scFv domain can be located in any orientation from the N-end to the C-end (VH-joint-VL or VL-joint-VH).

[0221] In this article, “single-domain Fv”, “sdFv”, or “sdABD” refers to an antigen-binding domain with only three CDRs, typically based on camel antibody technology. See: Protein Engineering 9(7): 1129-35 (1994); RevMol Biotech 74: 277-302 (2001); Ann Rev Biochem 82: 775-97 (2013). As outlined in this article, two general types of sdABD are used: sdABD bound to TTA and annotated therewith (sdABD-TTA for general terms, sdABD-EGFR for EGFR, and sdABD-FOLR1 for FOLR1) and sdABD bound to HSA (“sdABD-HSA” or “sdABD(1 / 2)”).

[0222] "Protein cleavage site" refers to the amino acid sequence that is recognized and cleaved by a protease. Suitable protease cleavage sites are summarized below and shown in Figures 5 and 6.

[0223] As used herein, "protease cleavage domain" refers to the term incorporated into "protease cleavage site" and other functional components of the construct of this invention, both between individual protease cleavage sites and at the protease cleavage site (e.g., V). H V L The peptide sequence of any linker between the iVL target antigen binding domain, half-life extension domain, etc. As described herein, the protease cleavage domain may also contain additional amino acids, for example, to impart flexibility.

[0224] The term "COBRA" TM "Conditional bispecific retargeting activation" refers to a conditionally effective protein with multiple functional protein domains. In some embodiments, one of the functional domains is an antigen-binding domain (ABD) that binds to a target tumor antigen (TTA). In some embodiments, another domain is an ABD that binds to a T-cell antigen under certain conditions. T-cell antigens include, but are not limited to, CD3. The term "half-cobra" is also used. TM"This refers to the innate self-assembly of variable heavy chains in hemi-cobras due to their concentration on the surface of target-expressing cells, allowing them to interact with another hemi-cobra." TM (Complementary COBRA) TM The variable light chain of a protein can bind conditionally to T-cell antigens when associated with the protein. Detailed Implementation

[0225] I. The fusion protein of the present invention

[0226] The fusion protein of the present invention has many different components (generally referred to herein as domains) linked together in a variety of ways. Some of these domains are binding domains, each binding to a target antigen (e.g., TTA or CD3). Because they bind to at most one antigen, they are referred to herein as “multispecific”; for example, the prodrug construct of the present invention can bind to both TTA and CD3, and is therefore “bispecific”. Proteins can also have higher specificity; for example, if a first αTTA binds to EGFR, a second αTTA binds to EpCAM, and an anti-CD3 binding domain is present, this would be a “trispecific” molecule. Similarly, adding an anti-HSA binding domain to this construct would make it “quadrispecific”, as... Figure 3B As shown in the image.

[0227] As those skilled in the art will understand, the proteins of the present invention may have different valences and are multispecific. That is, the proteins of the present invention may bind to targets having more than one binding site; for example, Pro140 is bivalent to EGFR.

[0228] The proteins of the present invention may include CD3 antigen-binding domains, tumor target antigen-binding domains, half-life extension domains, linkers, etc., arranged in a variety of ways as outlined herein.

[0229] A. CD3 antigen-binding domain

[0230] The specificity of T cell responses is mediated by the recognition of antigens by the T cell receptor complex (displayed against the backdrop of the major histocompatibility complex, MHC). As part of the T cell receptor complex, CD3 is a protein complex comprising the CD3γ (γ) chain, CD3δ (δ) chain, two CD3e (ε) chains, and two CD3ζ (ζ) chains present on the cell surface. The CD3 molecule associates with the α (α) and β (β) chains of the T cell receptor (TCR) to form the TCR complex. Clustering of CD3 on T cells (e.g., through binding to the Fv domain of CD3) leads to T cell activation, similar to T cell receptor conjugation, but independent of its clonal typical specificity.

[0231] However, as is known in the art, CD3 activation can cause numerous toxic side effects, and therefore, the present invention relates to providing active CD3 binding of the polypeptide of the invention only in the presence of tumor cells in which a specific protease is found, the protease then cleaving the prodrug polypeptide of the invention to provide an active CD3-binding domain. Thus, in the present invention, the binding of the anti-CD-3 Fv domain to CD-3 is regulated by a protease cleavage domain that restricts the binding of the CD-3 Fv domain to CD-3 only in the microenvironment of diseased cells or tissues with elevated protease levels (e.g., in the tumor microenvironment described herein).

[0232] Therefore, the present invention provides two sets of VH and VL domains, an active set (VH and VL) and an inactive set (iVH and iVL), all four of which are present in a prodrug construct. The construct is formalized such that the VH and VL sets do not self-associate but instead associate with inactive counterparts (e.g., iVH and VL and iVL and VH as shown herein).

[0233] 1. Active anti-CD3 variable heavy and light domains

[0234] Many suitable active CDR sets and / or VH and VL domains known in the art are applicable to the present invention. For example, the CDR and / or VH and VL domains are derived from known anti-CD-3 antibodies, such as moromuzumab-CD-3 (OKT3), osimertinib (TRX4), teplezumab (MGA031), vesizumab (Nuvion), SP34 or I2C, TR-66 or X35-3, VIT3, BMA030 (BW264 / 56), CLB-T3 / 3, and CRIS. 7. YTH12.5, F111-409, CLB-T3.4.2, TR-66, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87, 12F6, T3 / RW2-8C8, T3 / RW2-4B6, OKT3D, M-T301, SMC2, F101.01, UCHT-1, and WT-31.

[0235] In one embodiment, the VH and VL sequences forming the active Fv domain that binds to human CD3 are shown in Figure 5. As illustrated herein, these active VH (“aVH”) and active VL (“aVL”) domains can be used in different configurations and forms 1, 2, 3, and 4.

[0236] 2. Inactive anti-CD3 variable heavy and light domains

[0237] The inactive iVH and iVL domains contain “normal” frame regions (FRs) that allow association, such that the inactive variable domains will associate with the active variable domains, thereby rendering the pair inactive, for example, unable to bind CD3.

[0238] As those skilled in the art will understand, there are many “inactive” variable domains that can be used in this invention. Essentially, any variable domain having a human framework region can be used, which allows self-assembly with another variable domain, regardless of the amino acid at the CDR position in the variable region. For clarity, an inactive domain is referred to as containing a CDR, although technically an inactive variable domain does not confer binding ability.

[0239] As will be appreciated in the art, generating inactive VH or VL domains is generally straightforward and can be accomplished in a variety of ways. In some embodiments, the generation of inactive variable domains is typically accomplished by altering one or more CDRs of the active Fv, including altering one or more of the three CDRs of the active variable domain. This can be done by substituting one or more amino acid residues at functionally important residues in one or more CDRs, replacing some or all of the CDR residues with a random sequence, replacing one or more CDRs with a tag or label sequence, and / or exchanging CDRs and / or variable regions with CDRs and / or variable regions from unrelated antibodies (e.g., CDRs and / or variable regions of proteins targeting different organisms).

[0240] In some cases, only one of the CDRs in the variable region can be changed to make it inactive, although other implementations include changes in 1, 2, 3, 4, 5 or 6 CDRs.

[0241] In some cases, inactive domains can be engineered to promote selective binding in prodrug form to facilitate the formation of intramolecular iVH-VL and VH-iVL domains prior to cleavage (e.g., beyond intermolecular pair formation). See, for example, Igawa et al., Protein Eng. Des. Selection 23(8): 667-677 (2010), which is hereby expressly incorporated in its entirety by reference, and particularly with respect to amino acid substitutions at interfacial residues.

[0242] In some embodiments, the CD-3 binding domain of the peptide construct described herein not only exhibits efficient CD-3 binding affinity for human CD-3, but also demonstrates excellent cross-reactivity with the corresponding cynomolgus CD-3 protein. In some cases, the CD-3 binding domain of the peptide construct cross-reacts with CD-3 derived from cynomolgus monkeys. In some cases, the human:cynomolgus monkey KD ratio of CD-3 is between 5 and 0.2.

[0243] In some implementations, the CD-3 binding domain of the antigen-binding protein can be any domain that binds to CD-3, including but not limited to domains derived from monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, and humanized antibodies. In some cases, it is advantageous for the CD-3 binding domain to originate from the same species in which the antigen-binding protein will ultimately be used. For example, for use in humans, it may be advantageous for the CD-3 binding domain of the antigen-binding protein to contain human or humanized residues from the antigen-binding domain of an antibody or antibody fragment.

[0244] Therefore, in one aspect, the antigen-binding domain includes a humanized or human-binding domain. In one embodiment, the humanized or human anti-CD-3 binding domain includes one or more (e.g., all three) light chain complementarity-determining regions 1 (LC CDR1), 2 (LC CDR2), and 3 (LC CDR3) of the humanized or human anti-CD-3 binding domain described herein, and / or one or more (e.g., all three) heavy chain complementarity-determining regions 1 (HC CDR1), 2 (HC CDR2), and 3 (HC CDR3) of the humanized or human anti-CD-3 binding domain described herein, for example, a humanized or human anti-CD-3 binding domain comprising one or more (e.g., all three) LC CDRs and one or more (e.g., all three) HC CDRs.

[0245] In some embodiments, the humanized or human anti-CD-3 binding domain includes a CD-3-specific humanized or human light chain variable region, wherein the CD-3-specific light chain variable region comprises a human or non-human light chain CDR within a human light chain framework region. In some cases, the light chain framework region is a λ (λ) light chain framework. In other cases, the light chain framework region is a κ (κ) light chain framework.

[0246] In some embodiments, one or more CD-3 binding domains are humanized or fully human. In some embodiments, one or more activated CD-3 binding domains bind to CD-3 on cells expressing CD-3 at a KD of 1000 nM or less. In some embodiments, one or more activated CD-3 binding domains bind to CD-3 on cells expressing CD-3 at a KD of 100 nM or less. In some embodiments, one or more activated CD-3 binding domains bind to CD-3 on cells expressing CD-3 at a KD of 10 nM or less. In some embodiments, one or more CD-3 binding domains are cross-reactive with cynomolgus monkey CD-3. In some embodiments, one or more CD-3 binding domains comprise the amino acid sequence provided herein.

[0247] In some embodiments, the humanized or human anti-CD-3 binding domain includes a CD-3-specific humanized or human heavy chain variable region, wherein the CD-3-specific heavy chain variable region includes human or non-human heavy chain CDRs in the human heavy chain framework region.

[0248] In one embodiment, the anti-CD-3 binding domain is an Fv, the Fv comprising a light chain and a heavy chain having the amino acid sequence provided herein. In one embodiment, the anti-CD-3 binding domain comprises: a light chain variable region comprising at least one, two, or three modified (e.g., substituted), but no more than 30, 20, or 10 modified (e.g., substituted) amino acid sequences having the amino acid sequence of the light chain variable region provided herein, or a sequence having 95%-99% identity with the amino acid sequence provided herein; and / or a heavy chain variable region comprising at least one, two, or three modified (e.g., substituted), but no more than 30, 20, or 10 modified (e.g., substituted) amino acid sequences having the amino acid sequence of the heavy chain variable region provided herein, or a sequence having 95%-99% identity with the amino acid sequence provided herein. In one embodiment, the humanized or human anti-CD-3 binding domain is an scFv, and the light chain variable region comprising the amino acid sequence described herein is attached to the heavy chain variable region comprising the amino acid sequence described herein via an scFv linker. The light chain variable region and the heavy chain variable region of scFv can be, for example, in any of the following orientations: light chain variable region - scFv joint - heavy chain variable region or heavy chain variable region - scFv joint - light chain variable region.

[0249] In some embodiments, the CD-3 binding domain of the antigen-binding protein has an affinity for CD-3 on cells expressing CD3, with a KD of 1000 nM or less, 100 nM or less, 50 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 1 nM or less, or 0.5 nM or less. In some embodiments, the CD-3 binding domain of the antigen-binding protein has an affinity for CD-3ε, with a KD of 1000 nM or less, 100 nM or less, 50 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 1 nM or less, or 0.5 nM or less. In other embodiments, the CD-3 binding domain of the antigen-binding protein has a low affinity for CD-3, i.e., about 100 nM or greater.

[0250] The affinity for CD-3 can be determined, for example, by the ability of the antigen-binding protein itself or its CD-3-binding domain to bind to CD-3 when coated on an assay plate, displayed on the surface of microbial cells, or in solution, as is known in the art and typically measured using Biacore or Octet. The binding activity of the antigen-binding protein itself or its CD-3-binding domain to CD-3 can be determined by immobilizing the ligand (e.g., CD-3) or the antigen-binding protein itself or its CD-3-binding domain to beads, substrates, cells, etc. Reagents can be added to a suitable buffer, and the binding pair can be incubated at a given temperature for a period of time. After washing to remove unbound material, the bound protein can be released using, for example, SDS, a buffer with a high pH, ​​and analyzed, for example, by surface plasmon resonance (SPR).

[0251] In many embodiments, the preferred active and inert binding domains are those shown in Figure 5. Figure 5 depicts an active VH and VL, as well as three inactive VHi and three inactive VLi that have been inactivated in different ways.

[0252] As shown in Figure 5, a pair of particularly useful active anti-CD3VL and VH domains have VL, which has: vlCDR1 having SEQ ID NO: 127, vlCDR2 having SEQ ID NO: 128, and vlCDR3 having SEQ ID NO: 129; and VH, which has: vhCDR1 having SEQ ID NO: 143, vhCDR2 having SEQ ID NO: 144, and vhCDR3 having SEQ ID NO: 145.

[0253] As shown in Figure 5, a pair of particularly useful active anti-CD3VL and VH domains have VL, which has SEQ ID NO: 126; and VH, which has SEQ ID NO: 142.

[0254] B. Antigen-binding domain targeting tumor antigens

[0255] In addition to the described CD3 and half-life extension domains, the peptide constructs described herein also include target domains that bind to one or more regions on one or more target antigens or a single target antigen. It is contemplated herein that the peptide constructs of the present invention are cleaved at a protease cleavage domain, for example, in a disease-specific microenvironment or in the blood of a subject, and that each target antigen-binding domain binds to the target antigen on a target cell, thereby activating the CD3-binding domain to bind to T cells. Generally, TTA-binding domains may bind to their targets prior to protease cleavage, thus allowing them to “wait” on target cells to be activated as T cell adjuvants. At least one target antigen is involved in and / or associated with a disease, condition, or disorder. Exemplary target antigens include those associated with proliferative diseases, neoplastic diseases, inflammatory diseases, immune disorders, autoimmune diseases, infectious diseases, viral diseases, allergic reactions, parasitic reactions, graft-versus-host disease, or host-versus-graft disease. In some embodiments, the target antigen is a tumor antigen expressed on tumor cells. Or in some embodiments, the target antigen is associated with a pathogen, such as a virus or bacteria. At least one target antigen may also target healthy tissue.

[0256] In some embodiments, the target antigen is a cell surface molecule, such as a protein, lipid, or polysaccharide. In other embodiments, the target antigen is on tumor cells, virus-infected cells, bacterial-infected cells, damaged red blood cells, arterial plaque cells, or fibrotic tissue cells.

[0257] A preferred embodiment of the invention utilizes sdABD as the targeting domain. These are preferred over scFv ABD because adding other VH and VL domains to the constructs of the invention may complicate the formation of pseudo-Fv domains.

[0258] In some embodiments, the prodrug construct of the present invention utilizes a single TTA-binding domain, such as generally in Figure 3A Described as paired sdABD-TTA, and in Figure 4 Described as a "Form 4" configuration Figure 4 The use of a single anti-EGFR ABD is shown, although other TTA-binding domains may be used.

[0259] In some embodiments, particularly in constructs of forms 1 and 2, the prodrug constructs of the present invention utilize two TTA ABDs, again preferably in the form of sdABD-TTA. When dual targeting domains are used, they can bind to the same epitope of the same TTA. For example, as discussed herein, many constructs utilize two identical targeting domains. In some embodiments, two targeting domains binding to different epitopes of the same TTA can be used, for example, as shown in Figure 5, where two EGFR sdABDs bind to different epitopes on human EGFR. In some embodiments, two targeting domains bind to different TTAs, see, for example, a figure.

[0260] The polypeptide constructs considered herein include at least one antigen-binding domain, wherein said antigen-binding domain binds to at least one target antigen. In some embodiments, the target antigen-binding domain specifically binds to cell surface molecules. In some embodiments, the target antigen-binding domain specifically binds to a tumor antigen. In some embodiments, the target antigen-binding domain specifically and independently binds to a tumor target antigen (“TTA”) selected from at least one of EpCAM, EGFR, HER-2, HER-3, cMet, LyPD3, B7H3, CEA, Trop2, and FOLR1.

[0261] (a)EGFR sdABD

[0262] As shown in Figure 5, there are many particularly useful sdABDs that bind to human EGFR, which are referred to in this paper as "sdABD-EGFR" or "EGFRABD".

[0263] In one useful embodiment, sdABD-EGFR1 has: sdCDR1 having SEQ ID NO: 10, sdCDR2 having SEQ ID NO: 11, and sdCDR3 having SEQ ID NO: 12. In some cases, the sdABD-EGFR has SEQ ID NO: 9.

[0264] In one useful embodiment, sdABD-EGFR2a has: sdCDR1 having SEQ ID NO: 14, sdCDR2 having SEQ ID NO: 15, and sdCDR3 having SEQ ID NO: 16. In some cases, the sdABD-EGFR has SEQ ID NO: 13.

[0265] In one useful embodiment, sdABD-EGFR2d has: sdCDR1 having SEQ ID NO: 18, sdCDR2 having SEQ ID NO: 19, and sdCDR3 having SEQ ID NO: 20. In some cases, the sdABD-EGFR has SEQ ID NO: 17.

[0266] (b)EpCAM sdABD

[0267] As shown in Figure 5, there are many particularly useful sdABDs that bind to human EpCAM, referred to in this paper as "sdABD-EpCAM" or "EpCAMABD".

[0268] In one useful embodiment, sdABD-EpCAM h13 has: sdCDR1 having SEQ ID NO: 62, sdCDR2 having SEQ ID NO: 63, and sdCDR3 having SEQ ID NO: 64. In some cases, the sdABD-EpCAM has SEQ ID NO: 61.

[0269] In one useful embodiment, sdABD-EpCAM h23 has: sdCDR1 having SEQ ID NO: 66, sdCDR2 having SEQ ID NO: 67, and sdCDR3 having SEQ ID NO: 68. In some cases, the sdABD-EpCAM has SEQ ID NO: 65.

[0270] In a useful embodiment, sdABD-EpCAM hVIB665 has: sdCDR1 having SEQ ID NO: 70, sdCDR2 having SEQ ID NO: 71, and sdCDR3 having SEQ ID NO: 72. In some cases, the sdABD-EpCAM has SEQ ID NO: 69. It should be noted that, compared to h13 and h23EpCAM sdABD, hVIB665 (also known as "acEpCAM hVIB665") binds to both cleaved and uncleaved forms of EpCAM (which are known to undergo cleavage in vivo).

[0271] In a useful embodiment, sdABD-EpCAM hVIB666 has: sdCDR1 having SEQ ID NO: 74, sdCDR2 having SEQ ID NO: 75, and sdCDR3 having SEQ ID NO: 76. In some cases, the sdABD-EpCAM has SEQ ID NO: 73. It should be noted that, compared to h13 and h23 EpCAM sdABD, hVIB666 (also known as "acEpCAM hVIB666") binds to both cleaved and uncleaved forms of EpCAM (which are known to undergo cleavage in vivo).

[0272] (c)B7H3 sdABD

[0273] As shown in Figure 5, there are many particularly useful sdABDs that bind to human B7H3, which are referred to in this paper as "sdABD-B7H3" or "B7H3-ABD".

[0274] In one useful embodiment, sdABD-B7H3 hF7 has: sdCDR1 having SEQ ID NO: 34, sdCDR2 having SEQ ID NO: 35, and sdCDR3 having SEQ ID NO: 36. In some cases, sdABD-B7H3 has SEQ ID NO: 33.

[0275] In one useful embodiment, sdABD-B7H3 hF12 has: sdCDR1 having SEQ ID NO: 38, sdCDR2 having SEQ ID NO: 39, and sdCDR3 having SEQ ID NO: 40. In some cases, sdABD-B7H3 has SEQ ID NO: 37.

[0276] In one useful embodiment, sdABD-B7H3 hF12(N57Q) has: sdCDR1 having SEQ ID NO: 42, sdCDR2 having SEQ ID NO: 43, and sdCDR3 having SEQ ID NO: 44. In some cases, said sdABD-B7H3 has SEQ ID NO: 41. Compared to hF7 and hF12 B7H3 sdABD, the amino acid substitution N57Q removes the glycosylation site.

[0277] In one useful embodiment, sdABD-B7H3 HF12(N57E) has: sdCDR1 having SEQ ID NO: 46, sdCDR2 having SEQ ID NO: 47, and sdCDR3 having SEQ ID NO: 48. In some cases, said sdABD-B7H3 has SEQ ID NO: 45. Compared to hF7 and hF12 B7H3 sdABD, the amino acid substitution N57E removes the glycosylation site.

[0278] In one useful embodiment, sdABD-B7H3 hF12(N57D) has: sdCDR1 having SEQ ID NO: 50, sdCDR2 having SEQ ID NO: 51, and sdCDR3 having SEQ ID NO: 52. In some cases, said sdABD-B7H3 has SEQ ID NO: 49. Compared to hF7 and hF12 B7H3 sdABD, the amino acid substitution N57D removes the glycosylation site.

[0279] In one useful embodiment, sdABD-B7H3 hF12 (S59A) has: sdCDR1 having SEQ ID NO: 54, sdCDR2 having SEQ ID NO: 55, and sdCDR3 having SEQ ID NO: 56. In some cases, said sdABD-B7H3 has SEQ ID NO: 53. Compared to hF7 and hF12 B7H3 sdABD, the amino acid substitution S59A removes the glycosylation site.

[0280] In one useful embodiment, sdABD-B7H3 hF12(S59Y) has: sdCDR1 having SEQ ID NO: 58, sdCDR2 having SEQ ID NO: 59, and sdCDR3 having SEQ ID NO: 60. In some cases, said sdABD-B7H3 has SEQ ID NO: 57. Compared to hF7 and hF12 B7H3 sdABD, the amino acid substitution NS59Y removes the glycosylation site.

[0281] (d)FOLR1 sdABD

[0282] As shown in Figure 5, there are many particularly useful sdABDs that bind to human FOLR1, which are referred to in this paper as "sdABD-FOLR1" or "FOLR1-ABD".

[0283] In one useful embodiment, sdABD-FOLR1 h77-2 has: sdCDR1 having SEQ ID NO: 22, sdCDR2 having SEQ ID NO: 23, and sdCDR3 having SEQ ID NO: 24. In some cases, sdABD-FOLR1 has SEQ ID NO: 21.

[0284] In one useful embodiment, sdABD-FOLR1 h59.3 has: sdCDR1 having SEQ ID NO: 26, sdCDR2 having SEQ ID NO: 27, and sdCDR3 having SEQ ID NO: 28. In some cases, the sdABD-FOLR1 has SEQ ID NO: 25.

[0285] In one useful embodiment, sdABD-FOLR1 h22-4 has: sdCDR1 having SEQ ID NO: 30, sdCDR2 having SEQ ID NO: 31, and sdCDR3 having SEQ ID NO: 32. In some cases, sdABD-FOLR1 has SEQ ID NO: 29.

[0286] (e)Trop2 sdABD

[0287] As shown in Figure 5, there are many particularly useful sdABDs that bind to human Trop2, which are referred to in this paper as "sdABD-Trop2" or "Trop2-ABD".

[0288] In one useful implementation, sdABD-Trop2 hVIB557 has: sdCDR1 having SEQ ID NO: 78, sdCDR2 having SEQ ID NO: 79, and sdCDR3 having SEQ ID NO: 80. In some cases, sdABD-Trop2 has SEQ ID NO: 77.

[0289] In one useful implementation, sdABD-Trop2 hVIB565 has: sdCDR1 having SEQ ID NO: 82, sdCDR2 having SEQ ID NO: 83, and sdCDR3 having SEQ ID NO: 84. In some cases, sdABD-Trop2 has SEQ ID NO: 81.

[0290] In one useful implementation, sdABD-Trop2 hVIB575 has: sdCDR1 having SEQ ID NO: 86, sdCDR2 having SEQ ID NO: 87, and sdCDR3 having SEQ ID NO: 88. In some cases, sdABD-Trop2 has SEQ ID NO: 85.

[0291] In one useful implementation, sdABD-Trop2 hVIB578 has: sdCDR1 having SEQ ID NO: 90, sdCDR2 having SEQ ID NO: 01, and sdCDR3 having SEQ ID NO: 92. In some cases, the sdABD-Trop2 has SEQ ID NO: 89.

[0292] In one useful implementation, sdABD-Trop2 hVIB609 has: sdCDR1 having SEQ ID NO: 94, sdCDR2 having SEQ ID NO: 95, and sdCDR3 having SEQ ID NO: 96. In some cases, sdABD-Trop2 has SEQ ID NO: 93.

[0293] In one useful implementation, sdABD-Trop2 hVIB619 has: sdCDR1 having SEQ ID NO: 98, sdCDR2 having SEQ ID NO: 99, and sdCDR3 having SEQ ID NO: 100. In some cases, sdABD-Trop2 has SEQ ID NO: 97.

[0294] (f)CA9 sdABD

[0295] As shown in Figure 5, there are many particularly useful sdABDs that bind to human CA9, which are referred to in this paper as "sdABD-CA9" or "CA9-ABD".

[0296] In one useful embodiment, sdABD-CA9 hVIB456 has: sdCDR1 having SEQ ID NO: 102, sdCDR2 having SEQ ID NO: 103, and sdCDR3 having SEQ ID NO: 104. In some cases, sdABD-Trop2 has SEQ ID NO: 101.

[0297] In one useful embodiment, sdABD-CA9hVIB476 has: sdCDR1 having SEQ ID NO: 106, sdCDR2 having SEQ ID NO: 107, and sdCDR3 having SEQ ID NO: 108. In some cases, sdABD-Trop2 has SEQ ID NO: 105.

[0298] In one useful embodiment, sdABD-CA9 hVIB407 has: sdCDR1 having SEQ ID NO: 110, sdCDR2 having SEQ ID NO: 111, and sdCDR3 having SEQ ID NO: 112. In some cases, sdABD-Trop2 has SEQ ID NO: 109.

[0299] In one useful embodiment, sdABD-CA9 hVIB445 has: sdCDR1 having SEQ ID NO: 114, sdCDR2 having SEQ ID NO: 115, and sdCDR3 having SEQ ID NO: 116. In some cases, sdABD-Trop2 has SEQ ID NO: 113.

[0300] In some embodiments, the protein prior to cleavage of the protease cleavage domain is less than about 100 kDa. In some embodiments, the protein after cleavage of the protease cleavage domain is about 25 to about 75 kDa. In some embodiments, the protein prior to protease cleavage has a size higher than the renal threshold for first-pass clearance. In some embodiments, the protein prior to protease cleavage has an elimination half-life of at least about 50 hours. In some embodiments, the protein prior to protease cleavage has an elimination half-life of at least about 100 hours. In some embodiments, the protein has increased tissue penetration compared to IgG targeting the same target antigen. In some embodiments, the protein has increased tissue distribution compared to IgG targeting the same target antigen.

[0301] C. Half-life extension domain

[0302] The MCE protein of this invention (again referred to herein as "COBRA") TM The protein or construct may optionally include a half-life extension domain. Such domains include, but are not limited to, HSA-binding domains, Fc domains, small molecule domains, and other half-life extension domains known in the art.

[0303] Human serum albumin (HSA) (molecular weight approximately 67 kDa) is the most abundant protein in blood plasma, present at approximately 50 mg / ml (600 μM), and has a half-life of approximately 20 days in humans. HSA is used to maintain plasma pH, promote colloid blood pressure, act as a carrier for many metabolites and fatty acids, and serve as a major drug transport protein in plasma.

[0304] Non-covalent association with albumin prolongs the elimination half-life of short-lived proteins. For example, compared with administration of the Fab fragment alone, intravenous administration of the albumin-binding domain-Fab fragment resulted in a 25-fold and 58-fold decrease in in vivo clearance and a 26-fold and 37-fold prolongation of half-life in mice and rabbits, respectively. In another instance, when insulin was fatty acid-acylated to promote association with albumin, a durable effect was observed when administered subcutaneously to rabbits or pigs. These studies together demonstrate the link between albumin binding and prolongation of action.

[0305] In one embodiment, the antigen-binding protein described herein includes a half-life-extending domain, such as a domain that specifically binds to HSA. In other embodiments, the HSA-binding domain is a peptide. In other embodiments, the HSA-binding domain is a small molecule. Considering some embodiments, the HSA-binding domain of the antigen-binding protein is relatively small and does not exceed 25 kD, 20 kD, 15 kD, or 10 kD. In some cases, if the HSA-binding domain is a peptide or a small molecule, it is 5 kD or less.

[0306] In many implementations, the half-life extension domain is a single-domain antigen-binding domain derived from a single-domain antibody binding to HSA. This domain is generally referred to herein as “sdABD” (sdABD-HSA) for human HSA, or alternatively, “sdABD(1 / 2)”, to distinguish these binding domains from sdABD for TTA. A particularly useful sdABD(1 / 2) is shown in Figure 5.

[0307] The half-life extension domain of an antigen-binding protein provides altered pharmacodynamics and pharmacokinetics to the antigen-binding protein itself. As described above, the half-life extension domain extends the elimination half-life. The half-life extension domain also alters pharmacodynamic properties, including changing the tissue distribution, penetration, and diffusion of the antigen-binding protein. In some embodiments, the half-life extension domain provides improved tissue (including tumor) targeting, tissue penetration, tissue distribution, intra-tissue diffusion, and enhanced efficacy compared to proteins without the half-life extension domain. In one embodiment, the treatment method effectively and efficiently utilizes a reduced amount of antigen-binding protein, thereby producing reduced side effects, such as reduced cytotoxicity to non-tumor cells.

[0308] Furthermore, half-life-extending domains, such as HSA-binding domains, are characterized by their binding affinity for HSA. The affinity of the HSA-binding domain can be selected to target a specific elimination half-life in a particular peptide construct. Therefore, in some embodiments, the HSA-binding domain has high binding affinity. In other embodiments, the HSA-binding domain has moderate binding affinity. In still other embodiments, the HSA-binding domain has low or marginal binding affinity. Exemplary binding affinities include KD concentrations of 10 nM or less (high), between 10 nM and 100 nM (moderate), and greater than 100 nM (low). As described above, the binding affinity for HSA is determined using known methods such as surface plasmon resonance (SPR).

[0309] D. Protease cleavage site

[0310] As outlined herein, the protein compositions of the present invention, and particularly the prodrug constructs, contain one or more protease cleavage sites typically located in cleavable linkers.

[0311] As described herein, the prodrug constructs of the present invention comprise at least one protease cleavage site, said at least one protease cleavage site comprising an amino acid sequence cleaved by at least one protease. In some cases, the MCE protein described herein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more protease cleavage sites cleaved by at least one protease. As discussed more fully herein, when more than one protease cleavage site is used in a prodrug construct, said protease cleavage site may be the same (e.g., multiple sites cleaved by a single protease) or different (two or more cleavage sites cleaved by at least two different proteases). As those skilled in the art will understand, constructs containing three or more protease cleavage sites may utilize one, two, three, etc.; for example, some constructs may utilize three sites for two different proteases, etc.

[0312] The amino acid sequence of the protease cleavage site will depend on the protease being targeted. As is known in the art, there are many proteases that are found in vivo and can be associated with disease states.

[0313] Proteases are known to be secreted by some diseased cells and tissues, such as tumors or cancer cells, creating a protease-rich microenvironment. In some cases, the subject's blood is rich in proteases. In others, cells surrounding the tumor secrete proteases into the tumor microenvironment. Tumor-peritumor cells that secrete proteases include, but are not limited to, tumor stromal cells, myofibroblasts, blood cells, mast cells, B cells, NK cells, regulatory T cells, macrophages, cytotoxic T lymphocytes, dendritic cells, mesenchymal stem cells, polymorphonuclear cells, and other cells. In some cases, proteases are present in the subject's blood, such as proteases targeting amino acid sequences found in microbial peptides. This characteristic allows targeted therapeutic agents, such as antigen-binding proteins, to have additional specificity because T cells will not be bound by antigen-binding proteins except in the protease-rich microenvironment of the target cells or tissues.

[0314] Proteases are proteins that, under certain conditions, cleave proteins in a sequence-specific manner. Proteases include, but are not limited to, serine proteases, cysteine ​​proteases, aspartic proteases, threonine proteases, glutamate proteases, metalloproteinases, asparagine peptide lyases, serum proteases, cathepsins (e.g., cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K, cathepsin L, cathepsin S), kallikrein, hK1, hK10, hK15, KLK7, granzyme B, plasmin, collagenase, type IV collagenase, lysozyme, factor XA, chymotrypsin-like proteases, trypsin-like proteases, elastase-like proteases, subtilisin-like proteases, actinidin, bromelain, calpain, caspases (e.g., caspase-3), and Mir1-C. P, papain, HIV-1 protease, HSV protease, CMV protease, chymotrypsin, renin, pepsin, proteolytic enzyme, podase protease, Plasmodium aspartate protease, Nepenthesin, extrametallic peptidase, metalloendopeptidase, matrix metalloproteinase (MMP), MMP1, MMP2, MMP3, MMP8, MMP9, MMP13, MMP11, MMP14, Meprin, urokinase plasminogen activator (uPA), enterokinase, prostate-specific antigen (PSA, hK3), interleukin-1β convertase, thrombin, FAP (FAP-α), dipeptidyl peptidase, and dipeptidyl peptidase IV (DPPIV / CD26).

[0315] Some suitable proteases and protease cleavage sequences are shown in Figures 5 and 6.

[0316] E. Connector

[0317] As discussed herein, the different domains of the present invention are typically linked together using amino acid linkers, which may also impart functionality, including flexibility or rigidity (e.g., spatial constraint) and the ability to be cleaved by in situ proteases. These linkers can be classified in a variety of ways.

[0318] This invention provides "domain connectors" for linking two or more domains (e.g., VH and VL, a target tumor antigen binding domain (TTABD, sometimes referred to herein as "αTTA") (for "anti-TTA") to VH or VL, linking a half-life extension domain to another component, etc. Domain connectors can be non-cleavable (NCL), cleavable ("CL"), constrained and cleavable (CCL), and constrained and non-cleavable (CNCL), for example.

[0319] 1. Non-disintegrable connector

[0320] In some embodiments, the domain joints are non-cleavable. Generally, these can be of one of two types: non-cleavable and flexible, allowing the components “upstream” and “downstream” of the joint in the construct to self-assemble intramolecularly in some way; or non-cleavable and constrained, where the two components separated by the joint cannot self-assemble intramolecularly. However, it should be noted that in the latter case, although the two component domains separated by the non-cleavable, constrained joint do not self-assemble intramolecularly, other intramolecular components will self-assemble to form pseudo-Fv domains.

[0321] (i) Non-disintegrable but flexible joint

[0322] In this embodiment, the linker is used to connect the domains via a longer, flexible domain that is not cleaved by in situ proteases in the patient's body to preserve the functionality of the domains. Examples of cleavable linkers suitable for connecting the interior of domains in the peptides of the present invention include, but are not limited to, (GS)n, (GGS)n, (GGGS)n, (GGSG)n, (GGSGG)n, or (GGGGS)n, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the linker length may be about 15 amino acids.

[0323] (ii) Non-disintegrable and constrained joints

[0324] In some cases, the linker does not contain a cleavage site and is too short to allow intramolecular self-assembly of the protein domains separated by the linker, and is a “constrained non-cleavable linker” or “CNCL”. For example, in Pro186, the active VH and active VL are separated by 8 amino acids (“octamer”), which does not allow VH and VL to self-assemble into an active antigen-binding domain. In some embodiments, the linker remains flexible; for example, (GGGS)n, where n = 2. In other embodiments, although generally less preferred, more rigid linkers may be used, such as those comprising proline or bulky amino acids.

[0325] 2. Split-connector

[0326] All prodrug constructs described herein contain at least one cleavable linker. Therefore, in one embodiment, the domain linker is cleavable (CL), sometimes referred to herein as a “protease cleavage domain” (“PCD”). In this embodiment, the CL contains a protease cleavage site, as outlined herein and illustrated in Figures 5 and 6. In some cases, the CL contains only a protease cleavage site. Optionally, depending on the length of the cleavage recognition site, several additional linking amino acids may be present at one or both of the N-terminus or C-terminus of the CL. For example, 1, 2, 3, 4, or 5 amino acids may be present at one or both of the N-terminus and C-terminus of the cleavage site. Therefore, the cleavable linker can also be constrained (e.g., octamer) or flexible.

[0327] Of particular interest in this invention are MMP9 pyrolytic connectors and Meprin pyrolytic connectors, especially MMP9 constrained pyrolytic connectors and Meprin constrained pyrolytic connectors.

[0328] II. Structural domains of the present invention

[0329] This invention provides various forms of the prodrug peptides of this invention. This invention provides both constrained Fv domains and constrained pseudo-Fv domains. Additionally, this invention provides multivalent conditionally effective (“MCE”) proteins containing two Fv domains, but in non-isomerized constructs. As outlined herein, although each construct contains at least one protease-cleaving domain, these can be in a non-isomerized cleavable form or a non-isomerized non-cleavable form.

[0330] Importantly, although both types of domains (Fv domain and pseudo-Fv domain) are referred to as "constrained" in this paper, this means, as discussed above, that they are not constrained. Figure 36 , Figure 37 and Figure 38As shown, only one of these needs to be constrained, although generally, proteins are better expressed when both adapters are constrained.

[0331] Those skilled in the art will understand that, for forms 1, 2, and 4, there are four possibilities for the N-terminal to C-terminal order of the constrained and pseudo-Fv domains of the present invention (connectors not shown): aVH-aVL and iVL-iVH, aVH-aVL and iVH-iVL, aVL-aVH and iVL-iVH, and aVL-aVH and iVH-iVL. All four have been tested and all four are active, but the first order aVH-aVL and iVL-iVH has shown better expression than the other three. Therefore, although the description herein is generally shown in this aVH-aVL and iVL-iVH form, all disclosures herein also include other orders of these domains.

[0332] Note that, generally speaking, the N-terminus to C-terminus sequence of the full-length construct of the present invention is based on the aVH-aVL and iVL-iVH orientations.

[0333] Furthermore, it is known in the art that immunogenicity may exist in humans from C-terminal sequences derived from certain ABDs. Therefore, in general, especially when the C-terminus of the construct terminates in sdABD (e.g., the sdABD-HSA domain of many constructs), histidine tags (His6 or His10) can be used. For purification reasons, many or most sequences herein are generated using a His6 C-terminal tag; however, these sequences can also be used to reduce immunogenicity in humans, as illustrated by Holland et al., DOI 10.1007 / s10875-013-9915-0 and WO 2013 / 024059.

[0334] A. Constrained Fv domain

[0335] This invention provides a constrained Fv domain comprising an active VH and an active VL domain, which are covalently attached using constrained linkers (which, as outlined herein, can be cleavable (form 1) or non-cleavable (forms 2 and 4)). The constrained linkers prevent intramolecular association between aVH and aVL in the absence of cleavage. Therefore, the constrained Fv domain generally comprises a set of six CDRs contained within a variable domain, wherein vhCDR1, vhCDR2, and vhCDR3 of VH bind to CD-3, and vlCDR1, vlCDR2, and vlCDR3 of VL bind to CD-3; however, in a prodrug form (e.g., uncracked), VH and VL cannot spatially associate to form the active binding domain, but rather preferably pair intramolecularly with a pseudo-Fv.

[0336] As described herein, a constrained Fv domain may contain active VH and active VL (aVH and aVL) or inactive VH and VL (iVH and iVL, in which case it is a constrained pseudo-Fv domain) or a combination thereof.

[0337] As those skilled in the art will understand, in a constrained Fv structural domain, the order of VH and VL can be (N-end to C-end) VH-connector-VL or VL-connector-VH.

[0338] As outlined herein, for a Form 1 construct, the constrained Fv structural domain may comprise VH and VL connected using a detachable connector, as in those shown in Figures 5 and 6. In this embodiment, the constrained Fv structural domain has the structure (N-end to C-end) vhFR1-vhCDR1-vhFR2-vhCDR2-vhFR3-vhCDR3-vhFR4-CCL-vlFR1-vlCDR1-vlFR2-vlCDR2-vlFR3-vlCDR3-vlFR4. Generally, the constrained Fv domain contains active VH and VL domains (e.g., capable of binding CD3 upon association) and thus has the structure (N-terminus to C-terminus) vhFR1-avhCDR1-vhFR2-avhCDR2-vhFR3-avhCDR3-vhFR4-CCL-vlFR1-avlCDR1-vlFR2-avlCDR2-vlFR3-avlCDR3-vlFR4.

[0339] As outlined herein, for Form 2 constructs, the constrained Fv domain may comprise VH and VL connected using non-splitterable joints. In this embodiment, the constrained Fv domain has the structure (N-end to C-end) vhFR1-vhCDR1-vhFR2-vhCDR2-vhFR3-vhCDR3-vhFR4-CNCL-vlFR1-vlCDR1-vlFR2-vlCDR2-vlFR3-vlCDR3-vlFR4. Generally, the constrained Fv domain contains active VH and VL domains (e.g., capable of binding CD3 upon association) and thus has the structure (N-terminus to C-terminus) vhFR1-avhCDR1-vhFR2-avhCDR2-vhFR3-avhCDR3-vhFR4-CNCL-vlFR1-avlCDR1-vlFR2-avlCDR2-vlFR3-avlCDR3-vlFR4.

[0340] Particularly useful in this invention is the constrained, non-decomposable Fv domain having: aVH having SEQ ID NO: 142, aVL having SEQ ID NO: 126, and a domain connector having SEQ ID NO: 233.

[0341] B. Constrained pseudo-Fv domain

[0342] This invention provides a constrained pseudo-Fv domain comprising inactive or pseudo-iVH and iVL domains covalently attached using a constrained linker (which, as outlined herein, may be cleavable or non-cleavable). The constrained linker prevents intramolecular association between iVH and iVL in the absence of cleavage. Therefore, the constrained pseudo-Fv domain typically comprises iVH and iVL, having a framework region that allows iVH and iVL association (when in an unconstrained form), but the resulting pseudo-Fv domain does not bind to human proteins. The iVH domain can assemble with the aVL domain, and the iVL domain can assemble with the aVH domain, but the resulting structure does not bind to CD3.

[0343] The constrained pseudo-Fv domain contains inactive VH and VL (iVH and iVL).

[0344] As those skilled in the art will understand, in a constrained pseudo-Fv structural domain, the order of VH and VL can be (N-end to C-end) VH-joint-VL or VL-joint-VH.

[0345] As outlined in this paper, a constrained pseudo-Fv domain may contain iVH and iVL connected using non-splitter joints (as shown in Forms 1, 2 and 4) or splittable joints (as shown in Form 3).

[0346] Generally, the constrained Fv domain contains inert VH and VL domains (e.g., capable of binding CD3 upon association) and thus has the structure (N-terminus to C-terminus) vhFR1-ivlCDR1-vhFR2-ivlCDR2-vhFR3-ivlCDR3-vhFR4-CNCL-vlFR1-ivhCDR1-vlFR2-ivhCDR2-vlFR3-ivhCDR3-vlFR4.

[0347] Particularly useful in this invention is a constrained, non-decomposable pseudo-Fv domain having: an iVH having SEQ ID NO: 146, SEQ ID NO: 150 or SEQ ID NO: 154, an iVL having SEQ ID NO: 130, SEQ ID NO: 134 or SEQ ID NO: 138, and a domain connector having SEQ ID NO: 233.

[0348] III. Forms of the Invention

[0349] As discussed herein, the prodrug constructs of the present invention can take many different forms, including a cleavable form having two TTA-binding domains, a non-cleavable form having two TTA-binding domains (either of which may have the same TTA-binding domain or different binding domains), and a non-cleavable form having a single targeting domain.

[0350] A. A cleavable form with dual targeting

[0351] This invention is in Figure 1 The document provides a non-heterogeneous pyrolytic form of type "Form 1". In this embodiment, the constrained Fv domain comprises VH and VL domains connected using constrained pyrolytic joints, and the constrained pseudo-Fv domain uses constrained non-pyrolytic joints. For ease of discussion, both will be referred to herein as "constrained," but as discussed above and in [the context of the document], [the specific term is missing here]. Figure 37 , Figure 38 and Figure 39 As shown, only one of these needs to be constrained, although generally, proteins are better expressed when both adapters are constrained.

[0352] All constructs of form 1 (and others) also have a cleavable linker (CL) that can be cleaved by human tumor proteases.

[0353] The present invention provides a prodrug protein comprising, from the N-terminus to the C-terminus, (sdABD-TTA1)-domain linker-constrained Fv domain-domain linker-(sdABD-TTA2)-CL-constrained pseudo-Fv domain-domain linker-sdABD-HSA.

[0354] As those skilled in the art will understand, in a constrained Fv structural domain or a constrained pseudo-Fv structural domain, the order of VH and VL can be (N-end to C-end) VH-connector-VL or VL-connector-VH.

[0355] Therefore, in one embodiment, the prodrug protein comprises, from the N-terminus to the C-terminus: (sdABD-TTA1)-domain linker-aVH-CCL-aVL-domain linker-(sdABD-TTA2)-CL-iVL-CNCL-iVH-domain linker-sdABD-HSA.

[0356] Therefore, in one embodiment, the prodrug protein comprises, from the N-terminus to the C-terminus: (sdABD-TTA1)-domain linker-aVH-CCL-aVL-domain linker-(sdABD-TTA2)-CL-iVH-CCL-iVL-domain linker-sdABD-HSA.

[0357] Therefore, in one embodiment, the prodrug protein comprises, from the N-terminus to the C-terminus: (sdABD-TTA1)-domain linker-aVL-CCL-aVH-domain linker-(sdABD-TTA2)-CL-iVL-CCL-iVH-domain linker-sdABD-HSA.

[0358] Therefore, in one embodiment, the prodrug protein comprises, from the N-terminus to the C-terminus: (sdABD-TTA1)-domain linker-aVL-CCL-aVH-domain linker-(sdABD-TTA2)-CL-iVH-CCL-iVL-domain linker-sdABD-HSA.

[0359] In some embodiments, the prodrug construct comprises sdABD(TTA1)-domain connector-aVH-CCL-aVL-domain connector-sdABD(TTA2)-CL-iVL-CNCL-iVH-NCL-sdABD(1 / 2). In this embodiment, aVH, aVL, iVH, and iVL have the sequences shown in Figure 5.

[0360] In some embodiments, the prodrug construct comprises sdABD(TTA1)-domain connector-aVH-CCL-aVL-domain connector-sdABD(TTA2)-CL-iVL-CNCL-iVH-domain connector-sdABD(1 / 2). In this embodiment, aVH, aVL, iVH, and iVL have the sequences shown in Figure 5. In this embodiment, two targeting domains bind to the same TTA, which may be EGFR, EpCAM, FOLR1, Trop2, CA9, or B7H3, the sequence of which is depicted in Figure 5.

[0361] In some embodiments, the prodrug construct comprises sdABD(TTA1)-domain connector-aVH-CCL-aVL-domain connector-sdABD(TTA2)-CL-iVL-CNCL-iVH-domain connector-sdABD(1 / 2). In this embodiment, aVH, aVL, iVH, and iVL have the sequences shown in Figure 5. In this embodiment, the two targeting domains bind to different TTAs.

[0362] In some embodiments, the prodrug construct comprises sdABD(TTA1)-domain connector-aVH-CCL-aVL-domain connector-sdABD(TTA2)-CL-iVL-CNCL-iVH-domain connector-sdABD(1 / 2). In this embodiment, aVH, aVL, iVH, and iVL have the sequences shown in Figure 5. In this embodiment, the two targeting domains bind to EGFR and EpCAM, and sdABD-TTA has the sequences shown in Figure 5.

[0363] In some embodiments, the prodrug construct comprises sdABD(TTA1)-domain linker-aVH-CCL-aVL-domain linker-sdABD(TTA2)-CL-iVL-CNCL-iVH-domain linker-sdABD(1 / 2). In this embodiment, aVH, aVL, iVH, and iVL have the sequences shown in Figure 5. In this embodiment, the two targeting domains bind to EGFR and FOLR1, and sdABD-TTA has the sequences shown in Figure 5.

[0364] In some embodiments, the prodrug construct comprises sdABD(TTA1)-domain linker-aVH-CCL-aVL-domain linker-sdABD(TTA2)-CL-iVL-CNCL-iVH-domain linker-sdABD(1 / 2). In this embodiment, aVH, aVL, iVH, and iVL have the sequences shown in Figure 5. In this embodiment, the two targeting domains bind to EGFR and B7H3, and sdABD-TTA has the sequences shown in Figure 5.

[0365] In some embodiments, the prodrug construct comprises sdABD(TTA1)-domain connector-aVH-CCL-aVL-domain connector-sdABD(TTA2)-CL-iVL-CNCL-iVH-domain connector-sdABD(1 / 2). In this embodiment, aVH, aVL, iVH, and iVL have the sequences shown in Figure 5. In this embodiment, the two targeting domains bind to EpCAM and FOLR1, and sdABD-TTA has the sequences shown in Figure 5.

[0366] In some embodiments, the prodrug construct comprises sdABD(TTA1)-domain linker-aVH-CCL-aVL-domain linker-sdABD(TTA2)-CL-iVL-CNCL-iVH-domain linker-sdABD(1 / 2). In this embodiment, aVH, aVL, iVH, and iVL have the sequences shown in Figure 5. In this embodiment, the two targeting domains bind to EpCAM and B7H3, and sdABD-TTA has the sequences shown in Figure 5.

[0367] In some embodiments, the prodrug construct comprises sdABD(TTA1)-domain linker-aVH-CCL-aVL-domain linker-sdABD(TTA2)-CL-iVL-CNCL-iVH-domain linker-sdABD(1 / 2). In this embodiment, aVH, aVL, iVH, and iVL have the sequences shown in Figure 5. In this embodiment, the two targeting domains bind to B7H3 and FOLR1, and sdABD-TTA has the sequences shown in Figure 5.

[0368] In some embodiments, the prodrug construct comprises sdABD(TTA1)-domain linker-aVH-CCL-aVL-domain linker-sdABD(TTA2)-CL-iVL-CNCL-iVH-domain linker-sdABD(1 / 2). In this embodiment, aVH, aVL, iVH, and iVL have the sequences shown in Figure 5. In this embodiment, two targeting domains bind to the same TTA, which may be EGFR, FOLR1, B7H3, Trop2, CA9, or EpCAM, the sequence of which is depicted in Figure 5, and CCL and CL are selected from linkers cleaved by MMP9 or meprin, and sdABD(1 / 2) has SEQ ID NO: 117 or SEQ ID NO: 121.

[0369] In Form 1, the preferred structural domain connector is SEQ ID NO: 233 (which is also used as a preferred constrained, non-disintegrable connector).

[0370] In Form 1, the preferred constructs are Pro140 and Pro140b.

[0371] B. Indestructible form

[0372] like Figure 2As shown, the present invention provides a non-isomerized, non-cleavable form. In this embodiment, it should be understood that "non-cleavable" applies only to the binding of the constrained Fv domain, since an active cleavage site is present in the prodrug construct. In this embodiment, the constrained Fv domain comprises VH and VL domains connected using constrained, non-cleavable linkers, and the constrained pseudo-Fv domain uses constrained, non-cleavable linkers.

[0373] As those skilled in the art will understand, in a constrained Fv structural domain or a constrained pseudo-Fv structural domain, the order of VH and VL can be (N-end to C-end) VH-connector-VL or VL-connector-VH.

[0374] The present invention provides a prodrug protein comprising, from the N-terminus to the C-terminus, sdABD(TTA1)-domain linker-constrained Fv domain-domain linker-sdABD(TTA2)-cleavable linker-constrained pseudo-Fv domain-domain linker-sdABD-HSA.

[0375] As those skilled in the art will understand, in a constrained Fv structural domain or a constrained pseudo-Fv structural domain, the order of VH and VL can be (N-end to C-end) VH-connector-VL or VL-connector-VH.

[0376] Therefore, in one embodiment, the prodrug protein comprises, from the N-terminus to the C-terminus: (sdABD-TTA1)-domain linker-aVH-CNCL-aVL-domain linker-(sdABD-TTA2)-CL-iVL-CNCL-iVH-domain linker-sdABD-HSA.

[0377] Therefore, in one embodiment, the prodrug protein comprises, from the N-terminus to the C-terminus: (sdABD-TTA1)-domain linker-aVH-CNCL-aVL-domain linker-(sdABD-TTA2)-CL-iVH-CNCL-iVL-domain linker-sdABD-HSA.

[0378] Therefore, in one embodiment, the prodrug protein comprises, from the N-terminus to the C-terminus: (sdABD-TTA1)-domain linker-aVL-CNCL-aVH-domain linker-(sdABD-TTA2)-CL-iVL-CNCL-iVH-domain linker-sdABD-HSA.

[0379] Therefore, in one embodiment, the prodrug protein comprises, from the N-terminus to the C-terminus: (sdABD-TTA1)-domain linker-aVL-CNCL-aVH-domain linker-(sdABD-TTA2)-CL-iVH-CNCL-iVL-domain linker-sdABD-HSA.

[0380] In some embodiments, the prodrug protein comprises, from the N-terminus to the C-terminus: (sdABD-TTA1)-domain linker-aVH-CNCL-aVL-domain linker-(sdABD-TTA2)-CL-iVL-CNCL-iVH-domain linker-sdABD-HSA. In this embodiment, aVH, aVL, iVH, and iVL have the sequences shown in Figure 5. In this embodiment, two targeting domains bind to the same TTA, which may be EGFR, EpCAM, FOLR1, Trop2, CA9, or B7H3, the sequence of which is depicted in Figure 5.

[0381] In some embodiments, the prodrug protein comprises, from the N-terminus to the C-terminus: (sdABD-TTA1)-domain linker-aVH-CNCL-aVL-domain linker-(sdABD-TTA2)-CL-iVL-CNCL-iVH-domain linker-sdABD-HSA. In this embodiment, aVH, aVL, iVH, and iVL have the sequences shown in Figure 5. In this embodiment, the two targeting domains bind to different TTAs.

[0382] In some embodiments, the prodrug protein comprises, from the N-terminus to the C-terminus: (sdABD-TTA1)-domain linker-aVH-CNCL-aVL-domain linker-(sdABD-TTA2)-CL-iVL-CNCL-iVH-domain linker-sdABD-HSA. In this embodiment, aVH, aVL, iVH, and iVL have the sequences shown in Figure 5. In this embodiment, both targeting domains bind to EGFR and EpCAM, and sdABD-TTA has the sequences shown in Figure 5. In this embodiment, a preferred combination of EGFR and EpCAM includes:

[0383] cross EGFR2 EGFR2a EGFR2d EpCAM h13 any orientation any orientation any orientation EpCAM h23 any orientation any orientation any orientation EpCAM hVIB665 any orientation any orientation any orientation EpCAM hVIB666 any orientation any orientation any orientation

[0384] In this context, "any orientation" means that EpCAM sdABD is located at the N-terminus or C-terminus of EGFR sdABD in the construct of the present invention.

[0385] In some embodiments, the prodrug protein comprises, from the N-terminus to the C-terminus: (sdABD-TTA1)-domain linker-aVH-CNCL-aVL-domain linker-(sdABD-TTA2)-CL-iVL-CNCL-iVH-domain linker-sdABD-HSA. In this embodiment, aVH, aVL, iVH, and iVL have the sequences shown in Figure 5. In this embodiment, both targeting domains bind to EGFR and FOLR1, and sdABD-TTA has the sequences shown in Figure 5. In this embodiment, a preferred combination of EGFR and FOLR1 includes:

[0386] cross EGFR2 EGFR2a EGFR2d FOLR1h77-2 any orientation any orientation any orientation FOLR1 h59.3 any orientation any orientation any orientation FOLR h22-4 any orientation any orientation any orientation

[0387] In this context, "any orientation" means that the FORR1 sdABD is located at the N-terminus or the C-terminus of the EGFR sdABD in the construct of this invention.

[0388] In some embodiments, the prodrug protein comprises, from the N-terminus to the C-terminus: (sdABD-TTA1)-domain linker-aVH-CNCL-aVL-domain linker-(sdABD-TTA2)-CL-iVL-CNCL-iVH-domain linker-sdABD-HSA. In this embodiment, aVH, aVL, iVH, and iVL have the sequences shown in Figure 5. In this embodiment, both targeting domains bind to EGFR and B7H3, and sdABD-TTA has the sequences shown in Figure 5. In this embodiment, a preferred combination of EGFR and B7H3 includes:

[0389] cross EGFR2 EGFR2a EGFR2d B7H3 hF7 any orientation any orientation any orientation B7H3 hF12 any orientation any orientation any orientation B7H3 hF12(N57Q) any orientation any orientation any orientation B7H3 hF12(N57E) any orientation any orientation any orientation B7H3 hF12(N57D) any orientation any orientation any orientation B7H3 hF12(S59A) any orientation any orientation any orientation B7H3 hF12(S59Y) any orientation any orientation any orientation

[0390] In this context, "any orientation" means that B7H3 sdABD is located at the N-terminus or C-terminus of EGFR sdABD in the construct of this invention.

[0391] In some embodiments, the prodrug protein comprises, from the N-terminus to the C-terminus: (sdABD-TTA1)-domain linker-aVH-CNCL-aVL-domain linker-(sdABD-TTA2)-CL-iVL-CNCL-iVH-domain linker-sdABD-HSA. In this embodiment, aVH, aVL, iVH, and iVL have the sequences shown in Figure 5. In this embodiment, both targeting domains bind to EpCAM and FOLR1, and sdABD-TTA has the sequences shown in Figure 5. In this embodiment, a preferred combination of EpCAM and FOLR1 includes:

[0392] cross FOLR1h77-2 FOLR1 h59.3 FOLR h22-4 EpCAM h13 any orientation any orientation any orientation EpCAM h23 any orientation any orientation any orientation EpCAM hVIB665 any orientation any orientation any orientation EpCAM hVIB666 any orientation any orientation any orientation

[0393] In this context, "any orientation" means that EpCAM sdABD is located at the N-terminus or C-terminus of FORL1 sdABD in the construct of the present invention.

[0394] In some embodiments, the prodrug protein comprises, from the N-terminus to the C-terminus: (sdABD-TTA1)-domain linker-aVH-CNCL-aVL-domain linker-(sdABD-TTA2)-CL-iVL-CNCL-iVH-domain linker-sdABD-HSA. In this embodiment, aVH, aVL, iVH, and iVL have the sequences shown in Figure 5. In this embodiment, both targeting domains bind to EpCAM and B7H3, and sdABD-TTA has the sequences shown in Figure 5. In this embodiment, a preferred combination of EpCAM and B7H3 includes:

[0395]

[0396] In this context, "any orientation" means that B7H3 sdABD is located at the N-terminus or C-terminus of EGFR sdABD in the construct of this invention.

[0397] In some embodiments, the prodrug protein comprises, from the N-terminus to the C-terminus: (sdABD-TTA1)-domain linker-aVH-CNCL-aVL-domain linker-(sdABD-TTA2)-CL-iVL-CNCL-iVH-domain linker-sdABD-HSA. In this embodiment, aVH, aVL, iVH, and iVL have the sequences shown in Figure 5. In this embodiment, the two targeting domains bind to FOLR1 and B7H3, and sdABD-TTA has the sequences shown in Figure 5. In this embodiment, a preferred combination of FOLR1 and B7H3 includes:

[0398]

[0399]

[0400] In this context, "any orientation" means that B7H3 sdABD is located at the N-terminus or C-terminus of FORL1sdABD in the construct of this invention.

[0401] In some embodiments, the prodrug protein comprises, from the N-terminus to the C-terminus: (sdABD-TTA1)-domain linker-aVH-CNCL-aVL-domain linker-(sdABD-TTA2)-CL-iVL-CNCL-iVH-domain linker-sdABD-HSA. In this embodiment, aVH, aVL, iVH, and iVL have the sequences shown in Figure 5. In this embodiment, two targeting domains bind to the same TTA, which may be EGFR, FOLR1, B7H3, CA9, Trop2, or EpCAM, the sequence of which is depicted in Figure 5, and CCL and CL are selected from linkers cleaved by MMP9 or meprin, and sdABD(1 / 2) has SEQ ID NO: 117.

[0402] In Form 2, the preferred structural domain connector is SEQ ID NO: 233 (which is also used as a preferred constrained, non-disintegrable connector).

[0403] In Form 2, preferred dual-targeting constructs (sometimes referred to herein as “iso-COBRA”) include combinations targeting EGFR and EpCAM, EGFR and Trop2, EGFR and FORL1, EGF and B7H3, EpCAM and Trop2, EpCAM and FORL1, EpCAM and B7H3, Trop2 and FORL1, Trop2 and B7H3, and FORL1 and B7H3, as described more fully below.

[0404] In Form 2, the specific implementation schemes used include, but are not limited to, Pro186, Pro225, Pro226, Pro233, Pro262, Pro311, Pro312, Pro313, Pro356, Pro359, Pro364, Pro388, Pro448, Pro449, Pro450, Pro451, Pro495, Pro246, Pro254, Pro255, Pro256, Pro420, Pro421, Pro432, Pro479, Pro480, Pro187, Pro221, Pro222, Pro223, Pro224, Pro393, Pro394, Pro395, Pro396, Pro429, Pro430, Pro431, Pro601, Pro602, V3 and V4, Pro664, and Pro665. Pro667, Pro694, Pro695, Pro565, Pro566, Pro567, Pro727, Pro728, Pro729, Pro730, Pro731, Pro676, Pro677, Pro678, Pro679, Pro808, Pro819, Pro621, Pro622, Pro640, Pro641, Pro642, Pro643, Pro744, Pro746, Pro638, Pro639, Pro396, Pro476, Pro706, Pro709, Pro470, Pro471, Pro551, Pro552, Pro623, Pro624, Pro698, Pro655, Pro656, Pro657, Pro658, Pro516, Pro517, Pro518 and Pro519.

[0405] C. Single TTA construct

[0406] like Figure 4 As shown, the “Form 4” construct is also included in the compositions of the present invention, which is similar to the Form 2 construct but without the second TTA ABD. In this embodiment, it should be understood that “non-cleavable” applies only to the binding of the constrained Fv domain, as an active cleavage site is present in the prodrug construct. In this embodiment, the constrained Fv domain comprises VH and VL domains connected using a constrained non-cleavable linker, and the constrained pseudo-Fv domain uses a constrained non-cleavable linker.

[0407] As those skilled in the art will understand, in a constrained Fv structural domain or a constrained pseudo-Fv structural domain, the order of VH and VL can be (N-end to C-end) VH-connector-VL or VL-connector-VH.

[0408] This invention provides a prodrug protein comprising, from the N-terminus to the C-terminus, an sdABD(TTA)-domain linker-constrained Fv domain-cleavable linker-sdABD-HSA-constrained pseudo-Fv domain. (Note that for all constructs of this form, sdABD-HSA typically does not have a His6 tag, but may include one).

[0409] As those skilled in the art will understand, in a constrained Fv structural domain or a constrained pseudo-Fv structural domain, the order of VH and VL can be (N-end to C-end) VH-connector-VL or VL-connector-VH.

[0410] Therefore, in one embodiment, the prodrug protein comprises, from the N-terminus to the C-terminus: (sdABD-TTA)-domain linker-aVH-CNCL-aVL-CL-(sdABD-HSA)-domain linker-iVL-CNCL-iVH.

[0411] Therefore, in one embodiment, the prodrug protein comprises, from the N-terminus to the C-terminus: (sdABD-TTA)-domain linker-aVH-CNCL-aVL-CL-(sdABD-HSA)-domain linker-iVH-CNCL-iVL.

[0412] Therefore, in one embodiment, the prodrug protein comprises, from the N-terminus to the C-terminus: (sdABD-TTA)-domain linker-aVL-CNCL-aVH-CL-(sdABD-HSA)-domain linker-iVH-CNCL-iVL.

[0413] Therefore, in one embodiment, the prodrug protein comprises, from the N-terminus to the C-terminus: (sdABD-TTA)-domain linker-aVL-CNCL-aVH-CL-(sdABD-HSA)-domain linker-iVL-CNCL-iVH.

[0414] Therefore, in one embodiment, the prodrug protein comprises, from the N-terminus to the C-terminus: (sdABD-TTA)-domain linker-aVL-CNCL-aVH-CL-(sdABD-HSA)-domain linker-iVL-CNCL-iVH. In this embodiment, aVH, aVL, iVH, and iVL have the sequences shown in Figure 5. In this embodiment, the targeting domain binds to the TTA, which may be EGFR, EpCAM, FOLR1, Trop2, CA9, or B7H3, the sequence of which is depicted in Figure 5.

[0415] In Form 4, the preferred structural domain connector is SEQ ID NO: 233 (which is also used as a preferred constrained, non-disintegrable connector).

[0416] In form 4, the preferred sdABD-HSA is the sdABD-HSA of SEQ ID NO: 121 or 117.

[0417] D. A combination of two proteins

[0418] In some embodiments, the compositions of the present invention comprise two distinct molecules, sometimes referred to as "half-cobra". TM The term "or 'semi-construction'" refers to the intramolecular association of two distinct molecules to form a pseudo-Fv in the absence of cleavage. In the presence of a protease, the cleavage site is cleaved, thereby releasing an inert variable domain, and the protein pair then forms an active antigen-binding domain that binds to CD3, as generally depicted in Figure 3.

[0419] In the design of the semi-construct, it is important that the cleaved active variable domain and sdABD-TTA remain together so that the two cleaved parts are held together by the tumor antigen receptor on the tumor surface and can then form an active anti-CD3 binding domain.

[0420] There are two different general form 3 constructs, wherein each member in the pair has a single sdABD-TTA ( Figure 3A ) and those with two different sdABD-TTAs, each targeting a different TTA ( Figure 3B ).

[0421] 1. Semi-cobras with a single TTA-binding domain TM Constructor (Form 3A)

[0422] In some implementations, the first half-COBRA TM The N-terminus to the C-terminus has an sdABD(TTA1) domain connector - aVH-CL-iVL domain connector - sdABD(1 / 2), and the second has an sdABD(1 / 2)-domain connector - iVH-CL-aVL-domain connector - sdABD(TTA2). In this embodiment, aVH, aVL, iVH, iVL and sdABD(1 / 2) have the sequence shown in FIG5, and sdABD-TTAa binds to human EGFR, EpCAM, Trop2, CA9FOLR1 and / or B7H3, and has the sequence depicted in FIG5.

[0423] 2. Semi-cobra with dual TTA ABD TM Construct

[0424] In some implementations, each of the paired prodrug constructs may have two sdABD-TTA binding domains, such as Figure 3B As shown in the diagram. In this embodiment, the first member of the pair from the N-end to the C-end comprises sdABD-TTA1-structural domain connector-sdABD-TTA2-structural domain connector-aVH-CL-iVL-structural domain connector-sdABD(HAS), and the second member from the N-end to the C-end comprises sdABD-TTA1-structural domain connector-sdABD-TTA2-aVL-CL-iVH-structural domain connector-sdABD-HSA.

[0425] The two sdABD-TTAs on each member of the pair are different, but usually the two members (half-cobra) TM They all have the same two sdABD-TTAs, for example, they all have EGFR and FORR1 or EGFR and B7H3, etc.

[0426] In some implementations, the two sdABD-TTAs are selected from the sdABD-TTAs shown in Figure 5.

[0427] IV. Method for preparing the composition of the present invention

[0428] The prodrug compositions of the present invention are prepared as commonly understood by those skilled in the art and as outlined below.

[0429] This invention provides nucleic acid compositions encoding the prodrug compositions of this invention. As those skilled in the art will understand, the nucleic acid composition will depend on the form of the prodrug polypeptide. Thus, for example, when the form requires two amino acid sequences, such as the “form 3” construct, the two nucleic acid sequences can be incorporated into one or more expression vectors for expression. Similarly, prodrug constructs as single polypeptides (forms 1, 2, and 4) require a single nucleic acid in a single expression vector to be generated.

[0430] As is known in the art, nucleic acids encoding the components of the present invention can be incorporated into expression vectors as is known in the art, and depend on the host cells used to produce the prodrug compositions of the present invention. Generally, nucleic acids are operatively linked to any number of regulatory elements (promoters, origins of replication, selectivity markers, ribosome binding sites, inducers, etc.). The expression vector can be an extrachromosomal or integrated vector.

[0431] The nucleic acids and / or expression vectors of the present invention are then transformed into any number of different types of host cells known in the art, including mammalian, bacterial, yeast, insect and / or fungal cells, wherein mammalian cells (e.g., CHO cells, 293 cells) can be used in many embodiments.

[0432] As is well known in the art, the prodrug compositions of the present invention are prepared by culturing host cells containing the expression vector. Once generated, conventional antibody purification steps are performed, including protein A affinity chromatography and / or ion exchange chromatography.

[0433] V. Formulation and administration of the prodrug composition of the present invention

[0434] Formulations of the prodrug compositions used according to the invention are prepared for storage, in the form of lyophilized formulations or aqueous solutions, by mixing a prodrug of the desired purity (a single protein in the cases of forms 1, 2 and 4, and two proteins in the case of form 3) with an optional pharmaceutically acceptable carrier, excipient or stabilizer (as generally outlined in Remington's Pharmaceutical Sciences, 16th edition, Osol, A.Ed.

[1980] ).

[0435] The prodrug composition of the present invention is administered to a subject according to a known method, such as as a pill or by continuous intravenous infusion over a period of time.

[0436] The prodrug composition of the present invention can be used to treat cancer.

[0437] Example

[0438] A. Example 1: Construction and purification of the Pro construct

[0439] transfection

[0440] Each protein (e.g., single proteins of forms 1, 2, and 4) or paired constructs (form 3) was expressed from a single expression vector (pcdna3.4 derivative). Equal volumes of plasmid DNA encoding a pair of half-cobra or single-stranded constructs were mixed and transfected into Expi293 cells according to the manufacturer's transfection protocol. Five days post-transfection, the conditioned medium was harvested by centrifugation (6000 rpm x 25') and filtration (0.2 μM filter). Protein expression was confirmed by SDS-PAGE. The constructs were purified, and the final buffer composition was: 25 mM citrate, 75 mM arginine, 75 mM NaCl, 4% sucrose, pH 7. The final formulation was stored at -80°C.

[0441] MMP9 activation

[0442] Recombinant human (rh) MMP9 was activated according to the following protocol. Recombinant human MMP-9 (R&D#911-MP-010) was 0.44 mg / ml (4.7 μM). Phenylemamine para-aminoacetic acid (APMA) (Sigma) was prepared in DMSO at a stock concentration of 100 mM. The assay buffer consisted of 50 mM Tris pH 7.5, 10 mM CaCl2, 150 mM NaCl, and 0.05% Brij-35.

[0443] Dilute rhMMP9 to approximately 100 μg / ml with assay buffer (25 μL hMMP9 + 75 μL assay buffer).

[0444] Add p-aminoacetic acid phenylmercuric (APMA) to a final concentration of 1 mM (1 μL to 100 μL) from a 100 mM stock solution in DMSO.

[0445] -Incubate at 37°C for 24 hours

[0446] Dilute MMP9 to 10 ng / ul (add 900ul assay buffer to 100ul activation solution).

[0447] The concentration of activated rhMMP9 is approximately 100 nM.

[0448] Cleavage of the construct used for TDCC assay

[0449] To lyse the construct, supply CaCl2 to 10 mM in 100 μL of protein sample at a concentration of 1 mg / mL (10.5 μM) in preparation buffer (25 mM citrate, 75 mM L-arginine, 75 mM NaCl, 4% sucrose). Add activated rhMMP9 to a concentration of 20–35 nM. Incubate the sample overnight (16–20 h) at room temperature. Verify the integrity of the lysis using SDS-PAGE (10%–20% TG, TG run buffer, 200 v, 1 h). Samples are typically 98% lysed.

[0450] B. Example 2: T cell-dependent cytotoxicity (TDCC) assay

[0451] HT-29 cells transduced with firefly luciferase were grown to approximately 80% confluence and detached using Versene (0.48 mM EDTA-Ca-Mg in PBS). Cells were centrifuged and resuspended in TDCC medium (5% heat-inactivated FBS in RPMI 1640 containing HEPES, GlutaMax, sodium pyruvate, non-essential amino acids, and β-mercaptoethanol). Purified human P-pan-T cells were thawed, centrifuged, and resuspended in TDCC medium.

[0452] Cocultures of HT-29_Luc cells and T cells were added to 384-well cell culture plates. Serially diluted COBRA was then added to the cocultures and incubated at 37°C for 48 hours. Finally, an equal volume of SteadyGlo luciferase assay reagent was added to the plate and incubated for 20 minutes. The plates were read on a Perkin Elmer Envision with an exposure time of 0.1 s / well. Total fluorescence was read and data analyzed on a GraphPad Prism 7 or Type 8.3.1 (depending on timing).

[0453] C. Example 3: General Scheme Design for an In vivo Adoptive T-cell Transfer Efficacy Model

[0454] These protocols were used in many of the experiments shown in the figure. Tumor cells were subcutaneously (SC) implanted into the right abdomen of NSG (NOD.Cg-Prkdcscid Il2rgtm1Wj1 / SzJ) mice (The Jackson Laboratory, catalog number 005557) and allowed to grow until they reached an average volume of approximately 200 mm². 3 Tumors were established. Human T cells were cultured in parallel for approximately 10 days in T cell culture medium (X-VIVO 15 [Lonza, catalog 04-418Q], 5% human serum, 1% penicillin / streptomycin, 0.01 mM 2-mercaptoethanol) in a MACSiBead G-Rex 100M permeable flask (Wilson Wolf catalog 81100S) containing a T cell activation / expansion kit (Miltenyi catalog 130-091-441), supplemented with recombinant human IL-2 protein. Tumor growth in mice and activation / expansion of human T cells were coordinated so that on day 0 of the study, mice were randomly assigned to groups (N=6) based on tumor size; each mouse was then intravenously injected (IV) with 2.5 x 10⁻⁶ cells. 6 Mice were cultured human T cells and administered a first dose of either COBRA or a control molecule. Seven doses were administered to mice every 3 days (days 0, 3, 6, 9, 12, 15, and 18), and mice were then followed up for another 2–3 weeks until the tumor volume reached >2000 mmHg. 3 Or terminate the study. Measure tumor volume every 3 days.

[0455] D. Example 4: In vivo activity of EGFR / MMP9 hemi-COBRA against Pro77 and Pro53.

[0456] 5 x 10 6 1 LoVo cell or 5 x 10 6HT29 cells were subcutaneously implanted into the right abdomen of NSG (NOD.Cg-PrkdcscidIl2rgtm1Wjl / SzJ) mice (The Jackson Laboratory, catalog number 005557) and allowed to grow until tumor formation. Human T cells were cultured for 10 days in T cell medium (X-VIVO15 [Lonza, catalog number 04-418Q], 5% human serum, 1% penicillin / streptomycin, 0.01 mM 2-mercaptoethanol) in a MACSiBead G-Rex 100M vented flask (Wilson Wolf catalog number 81100S) containing a T cell activation / expansion kit (Miltenyi catalog number 130-091-441), supplemented with recombinant human IL-2 protein. To coordinate tumor growth in mice and activation / expansion of human T cells, mice were randomly assigned to groups (N=6) based on tumor size on day 0 of the study; each mouse was then intravenously injected (IV) with 2.5 x 10⁻⁶ cells. 6 Mice were cultured human T cells and administered a first dose of either COBRA or a control molecule. Seven doses were administered to mice every 3 days (days 0, 3, 6, 9, 12, 15, and 18), and mice were then monitored until tumor volume reached >2000 mm². 3 The study may be terminated if necessary. Each group received 0.2 mg / kg (mpk) of the anti-EGFR x CD3 positive control Pro51 bispecific antibody (bsAb), 0.5 mpk of the negative control anti-chicken egg lysozyme (HEL) x CD3 bsAb Pro98, 0.5 mpk of either the anti-EGFR hemi-cobra pairs Pro77 and Pro53 containing the MMP9 cleavable linker, or 0.5 mpk of either the anti-EGFR hemi-cobra pairs Pro74 and Pro72 containing the non-cleavable (NCL) linker. Tumor volume was measured every 3 days.

[0457] E. Example 5: In vivo activity of EGFR / MMP9 COBRA Pro140.

[0458] 5 x 10 6 1 LoVo cell or 5 x 10 6HT29 cells were subcutaneously implanted into the right abdomen of NSG (NOD.Cg-PrkdcscidIl2rgtm1Wj1 / SzJ) mice (The Jackson Laboratory, catalog number 005557) and allowed to grow until tumor formation. Human T cells were cultured in parallel for 10 days in T cell culture medium (X-VIVO15 [Lonza, catalog number 04-418Q], 5% human serum, 1% penicillin / streptomycin, 0.01 mM 2-mercaptoethanol) in a MACSiBead G-Rex 100M vented flask (Wilson Wolf catalog number 81100S) containing a T cell activation / expansion kit (Miltenyi catalog number 130-091-441), supplemented with recombinant human IL-2 protein. To coordinate tumor growth in mice and activation / expansion of human T cells, mice were randomly assigned to groups (N=6) based on tumor size on day 0 of the study; each mouse was then intravenously injected (IV) with 2.5 x 10⁻⁶ cells. 6 Mice were cultured human T cells and administered a first dose of either COBRA or a control molecule. Seven doses were administered to mice every 3 days (days 0, 3, 6, 9, 12, 15, and 18), and mice were then monitored until tumor volume reached >2000 mm². 3 The study may be terminated if necessary. Each group received 0.2 mpk of anti-EGFR x CD3 positive control Pro51 bispecific antibody (bsAb), 0.5 mpk of negative control anti-chicken egg lysozyme (HEL) x CD3 bsAb Pro98, or 0.5 mpk of anti-EGFR COBRAPro140 containing an MMP9 cleavable linker. Tumor volume was measured every 3 days.

[0459] F. Example 6: In vivo activity of EGFR / MMP9 COBRA Pro186.

[0460] 5 x 10 6HT29 cells were subcutaneously implanted into the right abdomen of NSG (NOD.Cg-Prkdcscid Il2rgtm1Wj1 / SzJ) mice (The Jackson Laboratory, catalog number 005557) and allowed to grow until tumor formation. Human T cells were cultured in parallel for 10 days in T cell culture medium (X-VIVO 15 [Lonza, catalog number 04-418Q], 5% human serum, 1% penicillin / streptomycin, 0.01 mM 2-mercaptoethanol) in a MACSiBead G-Rex 100M vented flask (Wilson Wolf catalog number 81100S) containing a T cell activation / expansion kit (Miltenyi catalog number 130-091-441), supplemented with recombinant human IL-2 protein. To coordinate tumor growth and human T cell activation / expansion in mice, mice were randomly assigned to groups (N=6) based on tumor size on day 0 of the study; each mouse was then intravenously injected with 2.5 x 10⁶ cultured human T cells and given a first dose of either COBRA or a control molecule. Mice were given seven doses every 3 days (days 0, 3, 6, 9, 12, 15, and 18), and then tracked until the tumor volume reached >2000 mm². 3 The study may be terminated if necessary. Each group received 0.1 mg / kg (mpk) of the anti-EGFR x CD3 positive control Pro51 bispecific antibody (bsAb), 0.3 mpk of anti-EGFR COBRA Pro214 containing a non-cleavable (NCL) control linker, 0.1 or 0.3 mpk of anti-EGFR COBRA Pro140 containing a cleavable MMP9 linker, or 0.1 or 0.3 mpk of anti-EGFR COBRA Pro186 containing a cleavable MMP9 linker. Tumor volume was measured every 3 days.

[0461] G. Example 7: Successful humanization of anti-EGFR sequences

[0462] The results are shown below.

[0463]

[0464] These results demonstrate that the humanization of the EGFR binding domain was successful, and that there is a strong affinity for the target EGFR when there are two binding sites on the molecule.

[0465] Example: Successful humanization of EpCAM sdABD

[0466] The results are shown below.

[0467]

[0468] These results demonstrate that the humanization of the EpCAM-binding domain was successful.

[0469] H. Example 8: COBRA TM A novel conditionally active bispecific antibody for resolving established solid tumors in mice.

[0470] Despite the clinical success of bispecific antibodies (bsAbs) targeting hematologic malignancies (e.g., bonnetumab, a CD19xCD3bsAb), efficacy in solid tumor indications remains a significant challenge. Because T-cell redirection bsAbs are so effective, even very low levels of target antigen expression on cell surfaces in normal tissues can quickly become a safety concern and severely limit the dose levels achievable by patients. This limits the likelihood of reaching effective concentrations and diminishes the therapeutic potential of these highly active molecules. Furthermore, identifying “clean” target antigens uniquely expressed on tumors rather than in normal tissues is extremely difficult at best.

[0471] To overcome these challenges, a technology called COBRA was developed. TM A novel recombinant bsAb platform (conditional bispecific redirection activation) COBRA is engineered to target a broader range of expressed and validated tumor cell surface antigens by concentrating T cell binding in the tumor microenvironment. The COBRA molecule is engineered to bind to a target antigen that is expressed on both tumor and normal cells but does not bind to T cells unless exposed to a proteolytic microenvironment, which is common in tumors but not in normal healthy tissues. Once bound to the tumor target antigen, protease-dependent linker cleavage allows COBRA to convert an inactive anti-CD3 scFv domain into an active CD3 scFv binding domain. After conversion, COBRA is then able to co-bind to both T cells and the target antigen simultaneously, thereby generating an effective cytolytic T cell response against tumor cells. Furthermore, COBRA is engineered with a half-life extension portion that is removed from the active molecule after proteolytic cleavage. This allows for continued circulation of inactive COBRA prior to tumor target binding and more rapid clearance of unbound active COBRA molecules, thereby reducing the likelihood of cytotoxic activity in normal tissues.

[0472] Here, a novel design of the COBRA molecule is revealed, and its ability to bind to CD3 and epidermal growth factor receptor (EGFR) to induce potent cytotoxic activity in T cell cultures and tumor-bearing mice implanted with human T cells is demonstrated. Low-to-subpicomolar T cell activation and cytotoxicity in vitro, as well as regression of established solid tumor xenografts in in vivo NSG mice mediated by COBRA adapter lysis-dependent T cells, have been reported.

[0473] Figures 64A-64CThe folding mechanism designed and predicted by COBRA is shown. Figure 64A A schematic diagram depicting PRO186 COBRA. Figure 64B The predicted COBRA fold is shown. The COBRA contains inactive VH and VL domains paired with anti-CD3 VH and VL domains. Uncleaved PRO186 COBRA binds to EGFR, CD3 binding is impaired, and it binds to serum albumin. Figure 64C The analytical size exclusion chromatogram of PRO186 is shown. The data indicate that unpyraminated PRO186 folds into a single structure.

[0474] Figures 65A-65C Exemplary embodiments of the constructs described herein include PRO186 (pre-cleaved PRO186), PRO186 cleavage products, and an active PRO186 dimer. One cleavage product comprises anti-CD3VH and VL domains and binds to EGFR with impaired CD3 binding. Another cleavage product comprises anti-CD3 inactive VH and VL domains and binds to serum albumin. The active PRO186 dimer comprises an active anti-CD3 agonist (a dimer of anti-CD3VH and VL) and binds to CD3 and EGFR.

[0475] Figure 66 Provides instructions on how COBRA is converted into an active dimer after protease cleavage.

[0476] Figures 67A-67B Provides characterization of COBRA binding. Figure 67A The study demonstrated binding activity against human, cynomolgus monkey, and mouse products. Figure 67B The binding of PRO186 to human CD3ε is shown; the activity of PRO186 binding to human CD3ε and the activity of PRO186 binding to human EGFR are also shown. Binding kinetics were assessed using Octet (Forte Bi) and EGFR (Acro Biosystems), serum albumin (Athens Research Technology), and CD3ε (Creative Biomart).

[0477] Figures 68A-68B The cleavage of the PRO186 connector by MMP2 and MMP9 is shown. Figure 68A To depict the protein imprint of the active binding product molecules after cleavage. Figure 68B This shows the cumulative effect of active binding product molecules relative to cleavage time.

[0478] Figure 69 The in vitro activity of the conditional PRO186 construct is shown. Figure 69 - The left figure shows the results of the T cell killing assay. Figure 69- The right figure shows the level of IFN-γ release in relation to the concentration of the test sample.

[0479] Figure 70 This study shows EGFR expression relative to activity in three tumor cell lines: LoVo (colorectal cancer (CRC) cell line), HT-29 (colorectal cancer (CRC) cell line), and SCC25 (head and neck cancer cell line). For in vitro EGFR expression, antibody / cell binding was measured using 1:1 PE-labeled anti-EGFR mAb#EGFR.1 and BD QuantiBrite Bead. For in vivo EGFR expression, IHC staining was performed using anti-EGFR mAb#WP84 and MACH4-HRP assay (Ensigna). For T cell killing assays, tumor cells expressing luciferase were co-cultured with human T cells at an E:T ratio of 10:1 for 48 hours and measured using Steady-Glo (Promega). For IFNγ release assays, IFNγ was measured at 24 hours at an E:T ratio of 10:1 using Meso ScaleDiscovery V-Pex.

[0480] Figure 71A and Figure 71B The expression of EGFR, MMP2, and MMP9 is shown in tumor cells and tumor xenografts. Figure 71A The surface density of EGFR cells is shown on three cancer cell lines—LoVo, HT-29, and SCC25. Figure 71B Immunohistochemical staining of EGFR, MMP2, and MMP9 in tumor xenografts is shown.

[0481] Figure 72 A schematic diagram of the experimental procedure for an adoptive human T-cell transfer model in tumor-bearing mice is provided. The experiment is used to measure in vivo antitumor efficacy and pharmacokinetics (PK). The procedure includes (1) subcutaneous implantation of a tumor in the right abdomen of an NSG mouse, (2) allowing the formation of an established tumor, such as a tumor of approximately 200 mm3, (3) administering q3dx7 to the mouse starting on day 0, (4) administering the final dose on day 18, and (5) terminating the study. The procedure also includes the following, performed concurrently with the in vivo experiment: (a) activating and expanding cultured human T cells for 10 days so that expansion begins at the same time as the tumor implantation process, and (b) harvesting T cells on day 0.

[0482] Figure 73 The study demonstrates the regression of solid tumors established in mice by PRO186. Figure 73 - The left image shows the regression of a LoVo-derived tumor. Figure 73 - The middle image shows the regression of a tumor derived from HT-29. Figure 73- The right figure shows the regression of tumors originating from SCC25.

[0483] Figures 74A-74B The results show that cleaved PRO186 is cleared faster than intact (uncleaved) PRO186. Figure 74A The pharmacokinetics of the test substance in the plasma of non-tumor-bearing mice are shown. Figure 74B The tumor volume of LoVo-derived tumors in mice that were administered the test sample is shown.

[0484] Conclusion: A multivalent sdAb-drug fusion compound was designed, which is converted into a highly effective bispecific T-cell redirection therapeutic agent upon proteolysis. In vitro assays showed that protease-dependent adaptor cleavage increased T-cell-mediated killing efficacy by 200-fold, resulting in a sub-picomolar therapeutic agent. Administration of PRO186 (Pro186) in mice with established xenografts led to protease-cleavage-dependent T-cell-mediated tumor regression in various tumor models. PRO186 demonstrated (1) a prolonged in vivo half-life after administration and (2) rapid clearance upon proteolytic activation, thus demonstrating that PRO186 is a therapeutic approach with improved safety profiles compared to conventional T-cell redirection bispecific drugs. sequence list <110> Takeda Pharmaceutical Co., Ltd. <120> Constrained conditionally activated binding proteins <130> 118459-5009-US, WO <160> 335 <170> PatentIn version 3.5 <210> 1 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> Synthetic antigen-binding domain-targeting sdAb -α-EGFR1 <400> 1 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Val Ala Ile Asn Trp Ser Ser Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Met Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Gly Tyr Gln Ile Asn Ser Gly Asn Tyr Asn Phe Lys Asp Tyr 100 105 110 Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 2 <211> 10 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> synthetic dCDR1 - target sdAb -α-EGFR1 <400> 2 Gly Arg Thr Phe Ser Ser Tyr Ala Met Gly 1 5 10 <210> 3 <211> 16 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> Synthesize sdCDR2 - target sdAb - α-EGFR1 <400> 3 Ile Asn Trp Ser Ser Gly Ser Thr Tyr Tyr Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 4 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Synthesize sdCDR3 - target sdAb - α-EGFR1 <400> 4 Gly Tyr Gln Ile Asn Ser Gly Asn Tyr Asn Phe Lys Asp Tyr Glu Tyr 1 5 10 15 Asp Tyr <210> 5 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> Synthetic antigen-binding domain-targeting sdAb -α-EGFR2 <400> 5 Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Ser Val Gln Thr Gly Gly 1 5 10 15 Ser Leu Arg Leu Thr Cys Ala Ala Ser Gly Arg Thr Ser Arg Ser Tyr 20 25 30 Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ser Gly Ile Ser Trp Arg Gly Asp Ser Thr Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Asp 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Ala Ala Ala Gly Ser Ala Trp Tyr Gly Thr Leu Tyr Glu Tyr Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 6 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthesize sdCDR1 - target sdAb - α-EGFR2 <400> 6 Gly Arg Thr Ser Arg Ser Tyr Gly Met Gly 1 5 10 <210> 7 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthesize sdCDR2 - target sdAb - α-EGFR2 <400> 7 Gly Ile Ser Trp Arg Gly Asp Ser Thr Gly Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 8 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthesize sdCDR3 - target sdAb - α-EGFR2 <400> 8 Ala Ala Gly Ser Ala Trp Tyr Gly Thr Leu Tyr Glu Tyr Asp Tyr 1 5 10 15 <210> 9 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> Synthetic antigen-binding domain-targeting sdAb - h-α-EGFR1 <400> 9 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Val Ala Ile Asn Trp Ser Ser Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Gly Tyr Gln Ile Asn Ser Gly Asn Tyr Asn Phe Lys Asp Tyr 100 105 110 Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 10 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthesize sdCDR1-targeting sdAb-h-α-EGFR1 <400> 10 Gly Arg Thr Phe Ser Ser Tyr Ala Met Gly 1 5 10 <210> 11 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthesize sdCDR2 - target sdAb - h-α-EGFR1 <400> 11 Ile Asn Trp Ser Ser Gly Ser Thr Tyr Tyr Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 12 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Synthesize sdCDR3 - target sdAb - h-α-EGFR1 <400> 12 Gly Tyr Gln Ile Asn Ser Gly Asn Tyr Asn Phe Lys Asp Tyr Glu Tyr 1 5 10 15 Asp Tyr <210> 13 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> Synthetic antigen-binding domain-targeting sdAb - aEGFR2a sdAb <400> 13 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Ser Arg Ser Tyr 20 25 30 Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ser Gly Ile Ser Trp Arg Gly Asp Ser Thr Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Ala Ala Ala Gly Ser Ala Trp Tyr Gly Thr Leu Tyr Glu Tyr Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 14 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthesize sdCDR1-targeted sdAb-aEGFR2a sdAb <400> 14 Gly Arg Thr Ser Arg Ser Tyr Gly Met Gly 1 5 10 <210> 15 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthesize sdCDR2 - target sdAb - aEGFR2a sdAb <400> 15 Gly Ile Ser Trp Arg Gly Asp Ser Thr Gly Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 16 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthesize sdCDR3 - target sdAb - aEGFR2a sdAb <400> 16 Ala Ala Gly Ser Ala Trp Tyr Gly Thr Leu Tyr Glu Tyr Asp Tyr 1 5 10 15 <210> 17 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> Synthetic antigen-binding domain - targeting sdAb - h-α-EGFR2 <400> 17 Gln Val Lys Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Gly 1 5 10 15 Ser Leu Thr Leu Ser Cys Ala Ala Ser Gly Arg Thr Ser Arg Ser Tyr 20 25 30 Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ser Gly Ile Ser Trp Arg Gly Asp Ser Thr Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Ala Ala Ala Gly Ser Ala Trp Tyr Gly Thr Leu Tyr Glu Tyr Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 18 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthesize sdCDR1 - target sdAb - h-α-EGFR2 <400> 18 Gly Arg Thr Ser Arg Ser Tyr Gly Met Gly 1 5 10 <210> 19 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthesize sdCDR2 - target sdAb - h-α-EGFR2 <400> 19 Gly Ile Ser Trp Arg Gly Asp Ser Thr Gly Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 20 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthesize sdCDR3 - target sdAb - h-α-EGFR2 <400> 20 Ala Ala Gly Ser Ala Trp Tyr Gly Thr Leu Tyr Glu Tyr Asp Tyr 1 5 10 15 <210> twenty one <211> 114 <212> PRT <213> Artificial Sequence <220> <223> Synthetic antigen-binding domain-targeting sdAb -α-FOLR1 h77-2 <400> twenty one Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Asn Ser 20 25 30 Val Met Ala Trp Tyr Arg Gln Thr Pro Gly Asn Glu Arg Glu Phe Val 35 40 45 Ala Ile Ile Asn Ser Ile Gly Ile Thr Asn Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Val Cys Asn 85 90 95 Arg Asn Phe Asp Arg Ile Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser <210> twenty two <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sdCDR1-targeting sdAb-α-FOLR1 h77-2 <400> twenty two Gly Phe Thr Val Ser Asn Ser Val Met Ala 1 5 10 <210> twenty three <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthesize sdCDR2 - target sdAb - α-FOLR1 h77-2 <400> twenty three Ile Ile Asn Ser Ile Gly Ile Thr Asn Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> twenty four <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sdCDR3-targeted sdAb-α-FOLR1 h77-2 <400> twenty four Asn Phe Asp Arg Ile Tyr 1 5 <210> 25 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Synthetic antigen-binding domain-targeting sdAb -α-FOLR1 h59.3 <400> 25 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Pro Gly Asn Thr Phe Ser Ile Ser 20 25 30 Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Trp Val 35 40 45 Ala Val Thr His Ser Asp Tyr Ser Thr Asn Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Lys 85 90 95 His Tyr Gly Ile Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110 Ser <210> 26 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sdCDR1 - targeted sdAb - α - FOLR1 h59.3 <400> 26 Gly Asn Thr Phe Ser Ile Ser Ala Met Gly 1 5 10 <210> 27 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthesized sdCDR2 - targeting sdAb - α-FOLR1 h59.3 <400> 27 Val Thr His Ser Asp Tyr Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 28 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthesized sdCDR3-targeted sdAb-α-FOLR1 h59.3 <400> 28 Tyr Gly Ile Asp Tyr 1 5 <210> 29 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Synthetic antigen-binding domain-targeting sdAb -α-FOLR1 h22-4 <400> 29 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Thr Thr Phe Ser Arg Asp 20 25 30 Val Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45 Ala Ile Ile Ser Arg Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Ala Asn Thr Ala Thr Trp Gly Arg Val Phe Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 30 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthesize sdCDR1-targeting sdAb-α-FOLR1 h22-4 <400> 30 Gly Thr Thr Phe Ser Arg Asp Val Met Gly 1 5 10 <210> 31 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthesize sdCDR2 - target sdAb - α-FOLR1 h22-4 <400> 31 Ile Ile Ser Arg Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 32 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthesize sdCDR3 - target sdAb - α-FOLR1 h22-4 <400> 32 Asn Thr Ala Thr Trp Gly Arg Val Phe 1 5 <210> 33 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> Synthetic antigen-binding domain-targeting sdAb -α-B7H3 hF7 <400> 33 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Pro Ser Arg Arg Thr Phe His Thr Tyr 20 25 30 His Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Val Ile Asn Trp Ser Gly Gly Ser Thr Val Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Gly Gly Ala Thr Thr Gln Arg Ala Thr Glu Ala Ser Tyr Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 34 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthesize sdCDR1 - target sdAb - α-B7H3 hF7 <400> 34 Arg Arg Thr Phe His Thr Tyr His Met Gly 1 5 10 <210> 35 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthesize sdCDR2 - target sdAb - α-B7H3 hF7 <400> 35 Val Ile Asn Trp Ser Gly Gly Ser Thr Val Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 36 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthesize sdCDR3 - target sdAb - α-B7H3 hF7 <400> 36 Gly Gly Ala Thr Thr Gln Arg Ala Thr Glu Ala Ser Tyr Asp Tyr 1 5 10 15 <210> 37 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Synthetic antigen-binding domain - targeting sdAb - α - B7H3 hF12 <400> 37 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ala Ser Pro Arg Thr Phe Ser Thr Tyr 20 25 30 Ser Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Ser Phe Val 35 40 45 Ala Ala Ile Asn Trp Ser Gly Gly Asn Thr Ser Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Gly Gly Val Leu Ala His His Asn Tyr Glu Tyr Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 38 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthesize sdCDR1 - target sdAb - α-B7H3 hF12 <400> 38 Pro Arg Thr Phe Ser Thr Tyr Ser Met Ala 1 5 10 <210> 39 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthesize sdCDR2 - target sdAb - α-B7H3 hF12 <400> 39 Ala Ile Asn Trp Ser Gly Gly Asn Thr Ser Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 40 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Synthesize sdCDR3 - target sdAb - α-B7H3 hF12 <400> 40 Gly Gly Val Leu Ala His His Asn Tyr Glu Tyr Asp Tyr 1 5 10 <210> 41 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Synthetic antigen-binding domain-targeting sdAb -α-B7H3 hF12 - (N57Q) <400> 41 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ala Ser Pro Arg Thr Phe Ser Thr Tyr 20 25 30 Ser Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Ser Phe Val 35 40 45 Ala Ala Ile Asn Trp Ser Gly Gly Gln Thr Ser Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Gly Gly Val Leu Ala His His Asn Tyr Glu Tyr Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 42 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthesize sdCDR1-targeting sdAb-α-B7H3 hF12-(N57Q) <400> 42 Pro Arg Thr Phe Ser Thr Tyr Ser Met Ala 1 5 10 <210> 43 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthesize sdCDR2 - targeting sdAb -α-B7H3 hF12 - (N57Q) <400> 43 Ala Ile Asn Trp Ser Gly Gly Gln Thr Ser Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 44 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Synthesize sdCDR3 - targeting sdAb -α-B7H3 hF12 - (N57Q) <400> 44 Gly Gly Val Leu Ala His His Asn Tyr Glu Tyr Asp Tyr 1 5 10 <210> 45 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Synthetic antigen-binding domain-targeting sdAb -α-B7H3 hF12 - (N57E) <400> 45 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ala Ser Pro Arg Thr Phe Ser Thr Tyr 20 25 30 Ser Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Ser Phe Val 35 40 45 Ala Ala Ile Asn Trp Ser Gly Gly Glu Thr Ser Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Gly Gly Val Leu Ala His His Asn Tyr Glu Tyr Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 46 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sdCDR1 - targeting sdAb - α - B7H3 hF12 - (N57E) <400> 46 Pro Arg Thr Phe Ser Thr Tyr Ser Met Ala 1 5 10 <210> 47 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthesize sdCDR2-targeting sdAb-α-B7H3 hF12-(N57E) <400> 47 Ala Ile Asn Trp Ser Gly Gly Glu Thr Ser Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 48 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Synthesize sdCDR3-targeting sdAb-α-B7H3 hF12-(N57E) <400> 48 Gly Gly Val Leu Ala His His Asn Tyr Glu Tyr Asp Tyr 1 5 10 <210> 49 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Synthetic antigen-binding domain-targeting sdAb -α-B7H3 hF12 - (N57D) <400> 49 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ala Ser Pro Arg Thr Phe Ser Thr Tyr 20 25 30 Ser Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Ser Phe Val 35 40 45 Ala Ala Ile Asn Trp Ser Gly Gly Asp Thr Ser Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Gly Gly Val Leu Ala His His Asn Tyr Glu Tyr Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 50 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sdCDR1 - targeting sdAb - α - B7H3 hF12 - (N57D) <400> 50 Pro Arg Thr Phe Ser Thr Tyr Ser Met Ala 1 5 10 <210> 51 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthesize sdCDR2-targeting sdAb-α-B7H3 hF12-(N57D) <400> 51 Ala Ile Asn Trp Ser Gly Gly Asp Thr Ser Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 52 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Synthesize sdCDR3-targeting sdAb-α-B7H3 hF12-(N57D) <400> 52 Gly Gly Val Leu Ala His His Asn Tyr Glu Tyr Asp Tyr 1 5 10 <210> 53 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Synthetic antigen-binding domain-targeting sdAb -α-B7H3 hF12 - (S59A) <400> 53 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ala Ser Pro Arg Thr Phe Ser Thr Tyr 20 25 30 Ser Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Ser Phe Val 35 40 45 Ala Ala Ile Asn Trp Ser Gly Gly Asn Thr Ala Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Gly Gly Val Leu Ala His His Asn Tyr Glu Tyr Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 54 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sdCDR1 - targeting sdAb - α - B7H3 hF12 - (S59A) <400> 54 Pro Arg Thr Phe Ser Thr Tyr Ser Met Ala 1 5 10 <210> 55 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthesize sdCDR2-targeting sdAb-α-B7H3 hF12-(S59A) <400> 55 Ala Ile Asn Trp Ser Gly Gly Asn Thr Ala Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 56 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Synthesize sdCDR3-targeting sdAb-α-B7H3 hF12-(S59A) <400> 56 Gly Gly Val Leu Ala His His Asn Tyr Glu Tyr Asp Tyr 1 5 10 <210> 57 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Synthetic antigen-binding domain-targeting sdAb -α-B7H3 hF12 - (S59Y) <400> 57 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ala Ser Pro Arg Thr Phe Ser Thr Tyr 20 25 30 Ser Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Ser Phe Val 35 40 45 Ala Ala Ile Asn Trp Ser Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Gly Gly Val Leu Ala His His Asn Tyr Glu Tyr Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 58 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthesized sdCDR1-targeting sdAb -α-B7H3 hF12 - (S59Y) <400> 58 Pro Arg Thr Phe Ser Thr Tyr Ser Met Ala 1 5 10 <210> 59 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthesize sdCDR2-targeting sdAb -α-B7H3 hF12 - (S59Y) <400> 59 Ala Ile Asn Trp Ser Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 60 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Synthesize sdCDR3-targeting sdAb-α-B7H3 hF12-(S59Y) <400> 60 Gly Gly Val Leu Ala His His Asn Tyr Glu Tyr Asp Tyr 1 5 10 <210> 61 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Synthetic antigen-binding domain-targeting sdAb -α-EpCAM h13 <400> 61 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Thr Leu Ser Cys Ala Ala Ser Gly Thr Gly Ser Ile Phe Ser 20 25 30 Ile Asn Leu Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu 35 40 45 Leu Val Ala Arg Ile Thr Ser Gly Asp Ser Thr Val Tyr Ala Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Asn Leu Leu Leu Arg Ser Ser Pro Gly Ala Thr Thr Pro Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 62 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Synthesize sdCDR1-targeted sdAb-α-EpCAM h13 <400> 62 Gly Thr Gly Ser Ile Phe Ser Ile Asn Leu Met Gly 1 5 10 <210> 63 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthesize sdCDR2 - target sdAb - α-EpCAM h13 <400> 63 Arg Ile Thr Ser Gly Asp Ser Thr Val Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 64 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Synthesize sdCDR3-targeted sdAb-α-EpCAM h13 <400> 64 Leu Leu Arg Ser Ser Pro Gly Ala Thr Thr Pro Tyr 1 5 10 <210> 65 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Synthetic antigen-binding domain-targeting sdAb -α-EpCAM h23 <400> 65 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Thr Leu Ser Cys Ala Ala Ser Gly Thr Gly Ser Ile Phe Ser 20 25 30 Ile Asn Leu Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu 35 40 45 Leu Val Ala Arg Ile Thr Ser Gly Asp Ser Thr Val Tyr Ala Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Asn Leu Leu Leu Arg Ser Ser Pro Gly Ala Thr Thr Pro Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 66 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthesize sdCDR1-targeting sdAb-α-EpCAM h23 <400> 66 Gly Ser Phe Ser Ala Leu Trp Ala Met Arg 1 5 10 <210> 67 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthesize sdCDR2 - target sdAb - α-EpCAM h23 <400> 67 Ser Ser Arg Gly Gly Thr Thr Ser Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 68 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthesize sdCDR3 - target sdAb - α-EpCAM h23 <400> 68 Ile Asp Gly His Leu Ala Tyr 1 5 <210> 69 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> Synthetic antigen-binding domain-targeting sdAb - acEpCAM hVIB665 <400> 69 Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Asp Tyr 20 25 30 Asp Met Gly Trp Phe Arg Gln Gly Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala Ile Ser Trp Ser Gly Gly His Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Asp Leu Arg Phe Thr Gly Gly Asp Thr Thr Thr Pro Glu Thr 100 105 110 Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 70 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sdCDR1 - targeting sdAb - acEpCAM hVIB665 <400> 70 Gly Arg Thr Phe Ser Asp Tyr Asp Met Gly 1 5 10 <210> 71 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sdCDR2 - Targeting sdAb - acEpCAM hVIB665 <400> 71 Ala Ile Ser Trp Ser Gly Gly His Thr Asn Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 72 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sdCDR3 - Targeted sdAb - acEpCAM hVIB665 <400> 72 Asp Leu Arg Phe Thr Gly Gly Asp Thr Thr Thr Pro Glu Thr Tyr Asp 1 5 10 15 Tyr <210> 73 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> Synthetic antigen-binding domain-targeting sdAb - acEpCAM hVIB666 <400> 73 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Leu Asp Asn Tyr 20 25 30 Asp Met Gly Trp Phe Arg Gln Gly Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala Ile Ser Trp Ser Gly Gly Ser Thr Asp Tyr Ala Tyr Ser Val 50 55 60 Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Ala Asp Leu Arg Phe Thr Gly Gly Asp Thr Met Thr Pro Glu Thr 100 105 110 Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 74 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sdCDR1-targeted sdAb - acEpCAM hVIB666 <400> 74 Gly Arg Thr Leu Asp Asn Tyr Asp Met Gly 1 5 10 <210> 75 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sdCDR2-targeted sdAb - acEpCAM hVIB666 <400> 75 Ala Ile Ser Trp Ser Gly Gly Ser Thr Asp Tyr Ala Tyr Ser Val Thr 1 5 10 15 Gly <210> 76 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sdCDR3-targeted sdAb - acEpCAM hVIB666 <400> 76 Asp Leu Arg Phe Thr Gly Gly Asp Thr Met Thr Pro Glu Thr Tyr Asp 1 5 10 15 Tyr <210> 77 <211> 129 <212> PRT <213> Artificial Sequence <220> <223> Synthetic antigen-binding domain - targeting sdAb - aTrop2 hVIB557 <400> 77 Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Gln 20 25 30 Ser Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ser Ala Ile Ser Trp Thr Gly Ala Asn Pro Thr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Asp Thr Ser Gly Gly Ser Tyr Tyr Tyr Glu Arg Ala Thr Ala 100 105 110 Glu Thr Ser Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115 120 125 Ser <210> 78 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sdCDR1-Targeting sdAb - aTrop2 hVIB557 <400> 78 Gly Arg Thr Phe Ser Ser Gln Ser Met Gly 1 5 10 <210> 79 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sdCDR2-targeted sdAb - aTrop2 hVIB557 <400> 79 Ala Ile Ser Trp Thr Gly Ala Asn Pro Thr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 80 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sdCDR3-targeted sdAb - aTrop2 hVIB557 <400> 80 Asp Thr Ser Gly Gly Ser Tyr Tyr Tyr Glu Arg Ala Thr Ala Glu Thr 1 5 10 15 Ser Tyr Asp Tyr 20 <210> 81 <211> 128 <212> PRT <213> Artificial Sequence <220> <223> Synthetic antigen-binding domain - targeting sdAb - aTrop2 hVIB565 <400> 81 Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Tyr Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ser Cys Ile Ser Ser Ser His Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Ala Gly Asp Gly Gly Asp Tyr His Cys Ser Gly Leu Val Asp 100 105 110 Tyr Gly Met Asp Tyr Trp Gly Lys Gly Thr Leu Val Thr Val Ser Ser [[ID=S29]]115 120 125 <210> 82 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sdCDR1-Targeting sdAb - aTrop2 hVIB565 <400> 82 Gly Phe Thr Phe Asp Tyr Tyr Ala Ile Gly 1 5 10 <210> 83 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sdCDR2-targeted sdAb - aTrop2 hVIB565 <400> 83 Cys Ile Ser Ser Ser His Gly Ser Thr Tyr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 84 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sdCDR3-targeted sdAb - aTrop2 hVIB565 <400> 84 Ala Gly Asp Gly Gly Asp Tyr His Cys Ser Gly Leu Val Asp Tyr Gly 1 5 10 15 Met Asp Tyr <210> 85 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> Synthetic antigen-binding domain - targeting sdAb - aTrop2 hVIB575 <400> 85 Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Leu Ala Ser Gly Arg Thr Val Gly Arg Thr 20 25 30 Ala Met Gly Trp Phe Arg Gln Pro Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Thr Ile Ser Trp Ala Gly Gly Thr Thr Tyr Tyr Ala Asp Phe Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Ser Glu Pro Tyr Ser Asp Tyr Asp Pro Ser Gly Met Val Tyr 100 105 110 Trp Gly Lys Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 86 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sdCDR1-Targeting sdAb - aTrop2 hVIB575 <400> 86 Gly Arg Thr Val Gly Arg Thr Ala Met Gly 1 5 10 <210> 87 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sdCDR2-targeted sdAb - aTrop2 hVIB575 <400> 87 Thr Ile Ser Trp Ala Gly Gly Thr Thr Tyr Tyr Ala Asp Phe Val Lys 1 5 10 15 Gly <210> 88 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sdCDR3-targeted sdAb - aTrop2 hVIB575 <400> 88 Ser Glu Pro Tyr Ser Asp Tyr Asp Pro Ser Gly Met Val Tyr 1 5 10 <210> 89 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> Synthetic antigen-binding domain - targeting sdAb - aTrop2 hVIB578 <400> 89 Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Gly Arg Ala 20 25 30 Ala Met Gly Trp Phe Arg Gln Pro Pro Gly Lys Glu Arg Glu Phe Ala 35 40 45 Ala Thr Ile Ser Trp Ser Gly Ser Asn Thr Tyr Tyr Ala Asp Phe Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Ser Glu Pro Tyr Ser Asp Tyr Asp Pro Ser Gly Met Val Tyr 100 105 110 Trp Gly Lys Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 90 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sdCDR1-targeted sdAb - aTrop2 hVIB578 <400> 90 Gly Arg Thr Phe Gly Arg Ala Ala Met Gly 1 5 10 <210> 91 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sdCDR2-targeted sdAb - aTrop2 hVIB578 <400> 91 Thr Ile Ser Trp Ser Gly Ser Asn Thr Tyr Tyr Ala Asp Phe Val Lys 1 5 10 15 Gly <210> 92 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sdCDR3-targeted sdAb - aTrop2 hVIB578 <400> 92 Ser Glu Pro Tyr Ser Asp Tyr Asp Pro Ser Gly Met Val Tyr 1 5 10 <210> 93 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Synthetic antigen-binding domain - targeting sdAb - aTrop2 hVIB609 <400> 93 Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Leu Ser Gly Leu Thr Phe Asn Thr Tyr 20 25 30 Pro Met Ala Trp Phe Arg Gln Pro Pro Gly Gln Glu Arg Glu Phe Val 35 40 45 Ala Asp Met Ser Trp Ser Gly Thr Asn Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Gly Trp Pro Tyr Ser Gly Thr Gly Arg Ser Thr Thr Asp Tyr 100 105 110 Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 94 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sdCDR1-targeted sdAb - aTrop2 hVIB609 <400> 94 Gly Leu Thr Phe Asn Thr Tyr Pro Met Ala 1 5 10 <210> 95 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sdCDR2-targeted sdAb - aTrop2 hVIB609 <400> 95 Asp Met Ser Trp Ser Gly Thr Asn Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 96 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sdCDR3-targeted sdAb - aTrop2 hVIB609 <400> 96 Gly Trp Pro Tyr Ser Gly Thr Gly Arg Ser Thr Thr Asp Tyr Thr Tyr 1 5 10 15 <210> 97 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> Synthetic antigen-binding domain - targeting sdAb - aTrop2 hVIB619 <400> 97 Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Ser Phe Ser Arg Tyr 20 25 30 Gly Met Gly Trp Leu Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val 35 40 45 Ala Ser Ile Ser Trp Ser Gly His Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Glu Ser Leu Pro Tyr Glu Ser Gly Ser Pro Arg Leu Thr Asp 100 105 110 Phe Ala Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 98 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sdCDR1-targeted sdAb - aTrop2 hVIB619 <400> 98 Gly Arg Ser Phe Ser Arg Tyr Gly Met Gly 1 5 10 <210> 99 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sdCDR2-targeted sdAb - aTrop2 hVIB619 <400> 99 Ser Ile Ser Trp Ser Gly His Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 100 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sdCDR3-targeted sdAb - aTrop2 hVIB619 <400> 100 Glu Ser Leu Pro Tyr Glu Ser Gly Ser Pro Arg Leu Thr Asp Phe Ala 1 5 10 15 Ser <210> 101 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Synthetic antigen-binding domain-targeting sdAb - aCA9 hVIB456 sdAb <400> 101 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ala Leu Ile Ile Asn 20 25 30 Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45 Ala Thr Val Thr Arg Ser Gly Arg Thr Asn Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Val Ala Leu Trp Ile Ala Asp Gly Glu Tyr Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 102 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sdCDR1-targeted sdAb - aCA9 hVIB456 sdAb <400> 102 Gly Ser Ala Leu Ile Ile Asn Ala Met Gly 1 5 10 <210> 103 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sdCDR2-targeted sdAb - aCA9 hVIB456 sdAb <400> 103 Thr Val Thr Arg Ser Gly Arg Thr Asn Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 104 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sdCDR3-targeted sdAb - aCA9 hVIB456 sdAb <400> 104 Ala Leu Trp Ile Ala Asp Gly Glu Tyr Asp Tyr 1 5 10 <210> 105 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Synthetic antigen-binding domain-targeting sdAb - aCA9 hVIB476 sdAb <400> 105 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asn Ile Phe Ile Ile Asn 20 25 30 Val Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45 Ala Thr Ile Thr Asn Gly Gly Arg Thr His Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Ala Asn His Ile Glu Leu Gly Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 106 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthesize sdCDR1-targeted sdAb - aCA9 hVIB476 sdAb <400> 106 Gly Asn Ile Phe Ile Ile Asn Val Met Gly 1 5 10 <210> 107 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthesize sdCDR2 - target sdAb - aCA9 hVIB476 sdAb <400> 107 Thr Ile Thr Asn Gly Gly Arg Thr His Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 108 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sdCDR3 - Targeted sdAb - aCA9 hVIB476 sdAb <400> 108 Asn His Ile Glu Leu Gly Asp Tyr 1 5 <210> 109 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Synthetic antigen-binding domain-targeting sdAb - aCA9 hVIB407 sdAb <400> 109 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Ile Ile Phe Ser Val Tyr 20 25 30 Asp Met Gly Trp Tyr Arg Gln Thr Pro Gly Lys Gln Arg Glu Phe Val 35 40 45 Ala Arg Ile Thr Ala Gly Gly Gly Thr Tyr Leu Thr Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Gly Val Tyr Tyr Cys Asn 85 90 95 Ala Ala Trp Ile Gly Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 110 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sdCDR1 - targeted sdAb - aCA9 hVIB407 sdAb <400> 110 Gly Ile Ile Phe Ser Val Tyr Asp Met Gly 1 5 10 <210> 111 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sdCDR2-targeted sdAb - aCA9 hVIB407 sdAb <400> 111 Arg Ile Thr Ala Gly Gly Gly Thr Tyr Leu Thr Asp Ser Val Lys Gly 1 5 10 15 <210> 112 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sdCDR3-targeted sdAb - aCA9 hVIB407 sdAb <400> 112 Ala Trp Ile Gly Asp Asp Tyr 1 5 <210> 113 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Synthetic antigen-binding domain-targeting sdAb - aCA9 hVIB445 sdAb <400> 113 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Asn Leu His 20 25 30 Ala Met Arg Trp Tyr Arg Arg Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45 Ala Tyr Ile Ser Ala Arg Asp Trp Thr Asn Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Thr Asp Leu Val Gly Glu Asp Tyr Trp Gly Arg Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 114 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthesize sdCDR1-targeted sdAb - aCA9 hVIB445 sdAb <400> 114 Gly Ile Thr Phe Asn Leu His Ala Met Arg 1 5 10 <210> 115 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthesize sdCDR2 - target sdAb - aCA9 hVIB445 sdAb <400> 115 Tyr Ile Ser Ala Arg Asp Trp Thr Asn Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 116 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sdCDR3 - Targeted sdAb - aCA9 hVIB445 sdAb <400> 116 Asp Leu Val Gly Glu Asp Tyr 1 5 <210> 117 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Synthetic antigen-binding domain-targeting sdAb -α-HSA Half-life extended structural domain (aHSA (10GE)) <400> 117 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Lys Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Arg Asp Thr Leu Tyr Ala Glu Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Val Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 118 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthesize the sdCDR1-targeted sdAb-α-HSA half-life extension domain (aHSA (10GE)). <400> 118 Gly Phe Thr Phe Ser Lys Phe Gly Met Ser 1 5 10 <210> 119 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthesize sdCDR2-targeted sdAb-α-HSA half-life extension domain (aHSA (10GE)) <400> 119 Ser Ile Ser Gly Ser Gly Arg Asp Thr Leu Tyr Ala Glu Ser Val Lys 1 5 10 15 Gly <210> 120 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthesize sdCDR3-targeted sdAb-α-HSA half-life extension domain (aHSA (10GE)) <400> 120 Gly Gly Ser Leu Ser Val 1 5 <210> 121 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Synthetic antigen-binding domain - α-HSA half-life extension domain <400> 121 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Lys Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Arg Asp Thr Leu Tyr Ala Glu Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Val Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser His His His His His 115 120 <210> 122 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sdCDR1 -α-HSA extended half-life domain <400> 122 Gly Phe Thr Phe Ser Lys Phe Gly Met Ser 1 5 10 <210> 123 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sdCDR2-α-HSA half-life extended domain <400> 123 Ser Ile Ser Gly Ser Gly Arg Asp Thr Leu Tyr Ala Glu Ser Val Lys 1 5 10 15 Gly <210> 124 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sdCDR3-α-HSA half-life extended domain <400> 124 Gly Gly Ser Leu Ser Val 1 5 <210> 125 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> Synthetic antigen-binding domain - α-CD3 scFv domain - α-CD3V(L) <400> 125 Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Ala Ser Ser Thr Gly Ala Val Thr Ser Gly 20 25 30 Asn Tyr Pro Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Lys Phe Leu Val Pro Gly Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Thr Leu Trp Tyr Ser Asn 85 90 95 Arg Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 126 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthesize the aVLCDR1 -αCD3scFV domain-α-CD3 V(L) <400> 126 Ala Ser Ser Thr Gly Ala Val Thr Ser Gly Asn Tyr Pro Asn 1 5 10 <210> 127 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthesize the aVLsdCDR2 -αCD3scFV domain-α-CD3 V(L) <400> 127 Gly Thr Lys Phe Leu Val Pro 1 5 <210> 128 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthesize the aVLsdCDR3 -αCD3scFV domain-α-CD3 V(L) <400> 128 Thr Leu Trp Tyr Ser Asn Arg Trp Val 1 5 <210> 129 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> Synthetic antigen-binding domain - α-CD3 scFv domain - α-CD3V(Li) <400> 129 Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Ser Gly 20 25 30 Asn Tyr Pro Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Asp Tyr Lys Asp Asp Asp Asp Lys Gly Thr Pro Ala Arg 50 55 60 Phe Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly 65 70 75 80 Val Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Val Leu Trp Tyr Ser 85 90 95 Asn Arg Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 130 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic iVLCDR1 - α CD3 scFV domain - α - CD3 V(Li) <400> 130 Gly Ser Ser Thr Gly Ala Val Thr Ser Gly Asn Tyr Pro Asn 1 5 10 <210> 131 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthesis of iVLsdCDR2 -αCD3scFV domain-α-CD3 V(Li) <400> 131 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 <210> 132 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthesis of iVLsdCDR3 -αCD3scFV domain-α-CD3 V(Li) <400> 132 Val Leu Trp Tyr Ser Asn Arg Trp Val 1 5 <210> 133 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> Synthetic antigen-binding domain - α-CD3 scFv domain - α-CD3V(Li2) <400> 133 Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Ser Gly 20 25 30 Asn Tyr Pro Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Lys Asp Asp Ala Pro Gly Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Val Leu Trp Tyr Ser Asn 85 90 95 Arg Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 134 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthesis of iVLCDR1 -αCD3scFV domain-α-CD3V(Li2) <400> 134 Gly Ser Ser Thr Gly Ala Val Thr Ser Gly Asn Tyr Pro Asn 1 5 10 <210> 135 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthesis of iVLCDR2 -αCD3scFV domain-α-CD3V(Li2) <400> 135 Gly Thr Lys Asp Asp Ala Pro 1 5 <210> 136 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthesis of iVLCDR3-αCD3scFV domain-α-CD3V(Li2) <400> 136 Val Leu Trp Tyr Ser Asn Arg Trp Val 1 5 <210> 137 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> Synthetic antigen-binding domain -α-CD3 scFv domain -α- CD3 VliGL <400> 137 Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Ser Gly 20 25 30 His Tyr Pro Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Ser Ser Asn Lys His Ser Trp Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Val Leu Trp Gly Ser Arg 85 90 95 Arg Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 138 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthesis of aVLiGLCDR1 -α-CD3 scFv domain -α-CD3 VliGL <400> 138 Gly Ser Ser Thr Gly Ala Val Thr Ser Gly His Tyr Pro Asn 1 5 10 <210> 139 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthesis of aVLiGLCDR2 -α-CD3 scFv domain -α-CD3 VliGL <400> 139 Gly Thr Ser Asn Lys His Ser 1 5 <210> 140 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthesis of aVLiGLCDR3 -α-CD3 scFv domain -α-CD3 VliGL <400> 140 Val Leu Trp Gly Ser Arg Arg Trp Val 1 5 <210> 141 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Synthetic antigen-binding domain - α-CD3 scFv domain - α-CD3V(H) <400> 141 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Tyr 20 25 30 Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Gln Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Ala Asn Phe Gly Asn Ser Tyr Ile Ser Tyr Trp 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 142 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthesis of aVHCDR1 -αCD3 scFV domain -α-CD3 V(H) <400> 142 Gly Phe Thr Phe Asn Lys Tyr Ala Ile Asn 1 5 10 <210> 143 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Synthesize the aVHsdCDR2 -αCD3scFV domain-α-CD3 V(H) <400> 143 Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Gln 1 5 10 15 Val Lys Asp <210> 144 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthesize the aVHsdCDR3 -αCD3scFV domain-α-CD3 V(H) <400> 144 His Ala Asn Phe Gly Asn Ser Tyr Ile Ser Tyr Trp Ala Tyr 1 5 10 <210> 145 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> Synthetic antigen-binding domain - α-CD3 scFv domain - α-CD3V(Hi) <400> 145 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asp Tyr Lys Asp Asp Asp Asp Lys Ala 50 55 60 Asp Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn 65 70 75 80 Thr Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val 85 90 95 Tyr Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Ile Ser Tyr 100 105 110 Trp Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 146 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic iVHsdCDR1 - α CD3 scFV domain - α - CD3 V(Hi) <400> 146 Gly Phe Thr Phe Asn Lys Tyr Ala Met Asn 1 5 10 <210> 147 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthesize the iVHsdCDR2 -αCD3 scFV domain -α-CD3 V(Hi). <400> 147 Arg Ile Arg Ser Lys Tyr Asp Tyr Lys Asp Asp Asp Asp Lys Ala Asp 1 5 10 15 Ser Val Lys Asp 20 <210> 148 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthesize the iVHsdCDR3 -αCD3 scFV domain -α-CD3 V(Hi). <400> 148 His Gly Asn Phe Gly Asn Ser Tyr Ile Ser Tyr Trp Ala Tyr 1 5 10 <210> 149 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Synthetic antigen-binding domain - α-CD3 scFv domain - α-CD3V(Hi2) <400> 149 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys His 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Ala Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Ile Ser Tyr Trp 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 150 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic iVHsdCDR1 - α CD3 scFV domain - α - CD3 V(Hi2) <400> 150 Gly Phe Thr Phe Asn Lys His Ala Met Asn 1 5 10 <210> 151 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Synthesize the iVHsdCDR2 -αCD3 scFV domain -α-CD3 V(Hi2). <400> 151 Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Ala Tyr Ala Asp Ser 1 5 10 15 Val Lys Asp <210> 152 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthesize the iVHsdCDR3 -αCD3 scFV domain -α-CD3 V(Hi2). <400> 152 His Gly Asn Phe Gly Asn Ser Tyr Ile Ser Tyr Trp Ala Tyr 1 5 10 <210> 153 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Synthetic antigen-binding domain -α-CD3 scFv domain -α - CD3 VHiGL4 <400> 153 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Ala Asn Ser Tyr Ala Thr Glu Tyr Ala Ala 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Ala Gly Asn Ser Ala Ile Ser Tyr Trp 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 154 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic aVHiGL4CDR1 - α - CD3 scFv domain - α - CD3 VHiGL4 <400> 154 Gly Phe Thr Phe Ser Gly Tyr Ala Met Asn 1 5 10 <210> 155 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Synthesis of aVHiGL4CDR2 -α-CD3 scFv domain -α-CD3 VHiGL4 <400> 155 Arg Ile Arg Ser Lys Ala Asn Ser Tyr Ala Thr Glu Tyr Ala Ala Ser 1 5 10 15 Val Lys Asp <210> 156 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthesis of aVHiGL4CDR3 -α-CD3 scFv domain -α-CD3 VHiGL4 <400> 156 His Gly Asn Ala Gly Asn Ser Ala Ile Ser Tyr Trp Ala Tyr 1 5 10 <210> 157 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic pyrolytic connector - MMP 2 / 9 <400> 157 Gly Pro Ala Gly Met Lys Gly Leu 1 5 <210> 158 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic pyrolytic connector - MMP 2 / 9 <400> 158 Ser Gly Gly Pro Gly Pro Ala Gly Met Lys Gly Leu Pro Gly Ser 1 5 10 15 <210> 159 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthetic pyrolytic connector - MMP 2 / 9 <400> 159 Ser Gly Gly Gly Pro Gly Pro Ala Gly Met Lys Gly Leu Pro Gly Gly 1 5 10 15 Ser <210> 160 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic pyrolytic connectors - Meprin A / B <400> 160 Gly Gly Gly Gly Lys Lys Leu Ala Asp Glu Pro Glu Gly Gly Gly Ser 1 5 10 15 <210> 161 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic pyrolytic connectors - Meprin A / B <400> 161 Ser Gly Gly Gly Lys Lys Leu Ala Asp Glu Pro Glu Gly Gly Ser 1 5 10 15 <210> 162 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic pyrolytic connectors - Meprin A / B <400> 162 Lys Lys Leu Ala Asp Glu Pro Glu 1 5 <210> 163 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Synthetic pyrolytic connector - Meprin A / B (variant, high efficiency) <400> 163 Gly Gly Gly Lys Phe Leu Ala Asp Glu Pro Glu Gly Gly 1 5 10 <210> 164 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthesize cleavable adaptors - cathepsins S, K, L <400> 164 Ser Gly Gly Gly Ala Arg Leu Gln Ser Ala Ala Pro Gly Gly Gly Ser 1 5 10 15 <210> 165 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthesize cleavable adaptors - cathepsins S, K, L <400> 165 Ser Gly Gly Gly Ala Arg Leu Gln Ser Ala Ala Pro Gly Gly Gly Ser 1 5 10 15 <210> 166 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthesize cleavable adaptors - cathepsins S, K, L <400> 166 Ala Arg Leu Gln Ser Ala Ala Pro 1 5 <210> 167 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Synthetic cleavable linker - Meprin / granulase B <400> 167 Ser Gly Gly Gly Gly Val Tyr Ala Asp Ser Leu Glu Asp Gly Gly Gly 1 5 10 15 Gly Ser <210> 168 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic cleavable linker - Meprin / granulase B <400> 168 Gly Val Tyr Ala Asp Ser Leu Glu Asp Gly 1 5 10 <210> 169 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthesize cleavable linker-protein lyase / uPA (MS) <400> 169 Gly Gly Gly Ser Leu Ser Gly Arg Ser Asp Asn His Gly Gly Gly Ser 1 5 10 15 <210> 170 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthesize cleavable linker-protein lyase / uPA (MS) <400> 170 Gly Leu Ser Gly Arg Ser Asp Asn His Gly 1 5 10 <210> 171 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthesize cleavable linker protein lysin (MV). <400> 171 Ser Gly Gly Gly Ser Phe Thr Arg Gln Ala Arg Val Val Gly Gly Gly 1 5 10 15 Ser <210> 172 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthesize cleavable linker protein lysin (MV). <400> 172 Ser Phe Thr Arg Gln Ala Arg Val Val 1 5 <210> 173 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthesize cleavable adaptors - cathepsin S / MMP9 / Meprin A <400> 173 Ala Arg Leu Gln Ser Ala Ala Pro Ala Gly Leu Lys Gly Ala 1 5 10 <210> 174 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthesize cleavable adaptors - cathepsin S / MMP9 / Meprin A <400> 174 Gly Ala Arg Leu Gln Ser Ala Ala Pro Ala Gly Leu Lys Gly Ala Gly 1 5 10 15 <210> 175 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Synthetic pyrolytic connector - MMP9 (variant, high efficiency) <400> 175 Gly Gly Pro Gly Pro Ala Gly Met His Gly Leu Pro Gly 1 5 10 <210> 176 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthetic pyrolytic connector - MMP9 (variant, high efficiency) <400> 176 Gly Ser Gly Gly Pro Gly Pro Ala Gly Met His Gly Leu Pro Gly Gly 1 5 10 15 Ser <210> 177 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Synthetic pyrolytic connector - MMP9 (variant, low efficiency) <400> 177 Gly Gly Pro Gly Pro Ala Gly Met Glu Gly Leu Pro Gly 1 5 10 <210> 178 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic pyrolytic connector - MMP9-15 (K>E) <400> 178 Ser Gly Gly Pro Gly Pro Ala Gly Met Glu Gly Leu Pro Gly Ser 1 5 10 15 <210> 179 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic pyrolytic connector - MMP9-15 (M>P) <400> 179 Ser Gly Gly Pro Gly Pro Ala Gly Pro Lys Gly Leu Pro Gly Ser 1 5 10 15 <210> 180 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic cleavable linker - thrombin1 <400> 180 Gly Gly Gly Gly Leu Val Pro Arg Gly Ser Leu Gly Gly Gly Gly Ser 1 5 10 15 <210> 181 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic cleavable linker - thrombin2 <400> 181 Ser Ser Gly Gly Gly Met Pro Arg Ser Phe Arg Gly Gly Gly Ser 1 5 10 15 <210> 182 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic cleavable linker-enterokinase / Flag <400> 182 Gly Gly Gly Gly Asp Tyr Lys Asp Asp Asp Asp Lys Gly Gly Gly Ser 1 5 10 15 <210> 183 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic pyrolytic connector - KLK7-6 <400> 183 Ser Gly Gly Gly Gln Asn Pro Tyr Ser Ala Gly Arg Gly Gly Gly Ser 1 5 10 15 <210> 184 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic pyrolytic connector - KLK7-13 <400> 184 Ser Gly Gly Gly Gln Asn Pro Tyr Ser Ala Gly Gly Gly Ser Gly Gly 1 5 10 15 <210> 185 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic pyrolytic connector - KLK7-11 <400> 185 Ser Gly Gly Gly Arg Asn Val Tyr Ser Ala Gly Gly Gly Ser Gly Gly 1 5 10 15 <210> 186 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic pyrolytic connector - KLK7-10 <400> 186 Ser Gly Gly Gly Gln Asn Thr Trp Ser Ala Gly Lys Gly Gly Gly Ser 1 5 10 15 <210> 187 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic pyrolytic connector - uPA <400> 187 Gly Gly Gly Ser His Thr Gly Arg Ser Ala Tyr Phe Gly Gly Gly Ser 1 5 10 15 <210> 188 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic pyrolytic connector - MMP9-2 <400> 188 Ser Gly Gly Pro Gly Pro Ala Gly Leu Lys Gly Ala Pro Gly Ser 1 5 10 15 <210> 189 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic protease cleavage site - MMP7 <400> 189 Lys Arg Ala Leu Gly Leu Pro Gly 1 5 <210> 190 <211> twenty four <212> PRT <213> Artificial Sequence <220> <223> Synthetic protease cleavage site - MMP7 <220> <221> MISC_FEATURE <222> (1)..(8) <223> X can be Asp or Glu <220> <221> MISC_FEATURE <222> (17) (24) <223> X can be Asp or Arg <400> 190 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg Pro Leu Ala Leu Trp Arg Ser 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 <210> 191 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic protease cleavage site - MMP9 <400> 191 Pro Arg Ser Thr Leu Ile Ser Thr 1 5 <210> 192 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic protease cleavage site - MMP9 <400> 192 Leu Glu Ala Thr Ala 1 5 <210> 193 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic protease cleavage site - MMP11 <400> 193 Gly Gly Ala Ala Asn Leu Val Arg Gly Gly 1 5 10 <210> 194 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic protease cleavage site - MMP14 <400> 194 Ser Gly Arg Ile Gly Phe Leu Arg Thr Ala 1 5 10 <210> 195 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic protease cleavage site - MMP <400> 195 Pro Leu Gly Leu Ala Gly 1 5 <210> 196 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic protease cleavage site - MMP <220> <221> misc_feature <222> (6)..(6) <223> Xaa can be any naturally occurring amino acid. <400> 196 Pro Leu Gly Leu Ala Xaa 1 5 <210> 197 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic protease cleavage site - MMP <220> <221> MISC_FEATURE <222> (5)..(5) <223> X can be Met or Glu <400> 197 Pro Leu Gly Cys Xaa Ala Gly 1 5 <210> 198 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic protease cleavage site - MMP <400> 198 Glu Ser Pro Ala Tyr Tyr Thr Ala 1 5 <210> 199 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic protease cleavage site - MMP <400> 199 Arg Leu Gln Leu Lys Leu 1 5 <210> 200 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic protease cleavage site - MMP <400> 200 Arg Leu Gln Leu Lys Ala Cys 1 5 <210> 201 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic protease cleavage sites - MMP2, MMP9, MMP14 <220> <221> MISC_FEATURE <222> (3)..(3) <223> X can be Cys, Ile, or Thr. <220> <221> MISC_FEATURE <222> (5)..(5) <223> X can be His, Orn, or Phe <400> 201 Glu Pro Xaa Gly Xaa Tyr Leu 1 5 <210> 202 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic protease cleavage site - urokinase plasminogen activator (UPA) <400> 202 Ser Gly Arg Ser Ala 1 5 <210> 203 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Synthetic protease cleavage site - urokinase plasminogen activator (UPA) <400> 203 Asp Ala Phe Lys 1 <210> 204 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic protease cleavage site - urokinase plasminogen activator (UPA) <400> 204 Gly Gly Gly Arg Arg 1 5 <210> 205 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Synthetic protease cleavage site - lysosomal enzyme <400> 205 Gly Phe Leu Gly 1 <210> 206 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Synthetic protease cleavage site - lysosomal enzyme <400> 206 Ala Leu Ala Leu 1 <210> 207 <211> 2 <212> PRT <213> Artificial Sequence <220> <223> Synthetic protease cleavage site - lysosomal enzyme <400> 207 Phe Lys 1 <210> 208 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic protease cleavage site - cathepsin B <400> 208 Asn Leu Leu 1 <210> 209 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic protease cleavage site - cathepsin D <220> <221> MISC_FEATURE <222> (4)..(4) <400> 209 Pro Ile Cys Xaa Phe Phe 1 5 <210> 210 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic protease cleavage site - cathepsin K <400> 210 Gly Gly Pro Arg Gly Leu Pro Gly 1 5 <210> 211 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic protease cleavage site - prostate-specific antigen <400> 211 His Ser Ser Lys Leu Gln 1 5 <210> 212 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic protease cleavage site - prostate-specific antigen <400> 212 His Ser Ser Lys Leu Gln Leu 1 5 <210> 213 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic protease cleavage site - prostate-specific antigen <400> 213 His Ser Ser Lys Leu Gln Glu Asp Ala 1 5 <210> 214 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic protease cleavage site - herpes simplex virus protease <400> 214 Leu Val Leu Ala Ser Ser Ser Phe Gly Tyr 1 5 10 <210> 215 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic protease cleavage site - Hiv protease <400> 215 Gly Val Ser Gln Asn Tyr Pro Ile Val Gly 1 5 10 <210> 216 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic protease cleavage site - Cmv protease <400> 216 Gly Val Val Gln Ala Ser Cys Arg Leu Ala 1 5 10 <210> 217 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Synthetic protease cleavage site - thrombin <220> <221> misc_feature <222> (2)..(2) <223> Xaa can be any naturally occurring amino acid. <400> 217 Phe Xaa Arg Ser 1 <210> 218 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic protease cleavage site - thrombin <400> 218 Asp Pro Arg Ser Phe Leu 1 5 <210> 219 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic protease cleavage site - thrombin <400> 219 Pro Pro Arg Ser Phe Leu 1 5 <210> 220 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Synthetic protease cleavage site - caspase-3 <400> 220 Asp Glu Val Asp 1 <210> 221 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic protease cleavage site - caspase-3 <400> 221 Asp Glu Val Asp Pro 1 5 <210> 222 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic protease cleavage site - caspase-3 <400> 222 Lys Gly Ser Gly Asp Val Glu Gly 1 5 <210> 223 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic protease cleavage site - interleukin-1-β convertase <400> 223 Gly Trp Glu His Asp Gly 1 5 <210> 224 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic protease cleavage site - enterokinase <400> 224 Glu Asp Asp Asp Asp Lys Ala 1 5 <210> 225 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic protease cleavage site - Fap <400> 225 Lys Gln Glu Gln Asn Pro Gly Ser Thr 1 5 <210> 226 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic protease cleavage site - kallikrein 2 <400> 226 Gly Lys Ala Phe Arg Arg 1 5 <210> 227 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Synthetic protease cleavage site - plasmin <400> 227 Asp Ala Phe Lys 1 <210> 228 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Synthetic protease cleavage site - plasmin <400> 228 Asp Val Leu Lys 1 <210> 229 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Synthetic protease cleavage site - plasmin <400> 229 Asp Ala Phe Lys 1 <210> 230 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic protease cleavage site - Top <400> 230 Ala Leu Leu Leu Ala Leu Leu 1 5 <210> 231 <211> 889 <212> PRT <213> Artificial Sequence <220> <223> Synthesized Construct - Pro140 Form 1 <400> 231 Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Ser Val Gln Thr Gly Gly 1 5 10 15 Ser Leu Arg Leu Thr Cys Ala Ala Ser Gly Arg Thr Ser Arg Ser Tyr 20 25 30 Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ser Gly Ile Ser Trp Arg Gly Asp Ser Thr Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Asp 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Ala Ala Ala Gly Ser Ala Trp Tyr Gly Thr Leu Tyr Glu Tyr Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Ser 115 120 125 Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val 130 135 140 Gln Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr 145 150 155 160 Phe Asn Lys Tyr Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly 165 170 175 Leu Glu Trp Val Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr 180 185 190 Tyr Tyr Ala Asp Gln Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp 195 200 205 Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp 210 215 220 Thr Ala Val Tyr Tyr Cys Val Arg His Ala Asn Phe Gly Asn Ser Tyr 225 230 235 240 Ile Ser Tyr Trp Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 245 250 255 Ser Gly Pro Ala Gly Met Lys Gly Leu Gln Thr Val Val Thr Gln Glu 260 265 270 Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Ala 275 280 285 Ser Ser Thr Gly Ala Val Thr Ser Gly Asn Tyr Pro Asn Trp Val Gln 290 295 300 Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr Lys Phe 305 310 315 320 Leu Val Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly 325 330 335 Lys Ala Ala Leu Thr Leu Ser Gly Val Gln Pro Glu Asp Glu Ala Glu 340 345 350 Tyr Tyr Cys Thr Leu Trp Tyr Ser Asn Arg Trp Val Phe Gly Gly Gly 355 360 365 Thr Lys Leu Thr Val Leu Gly Gly Gly Ser Gly Gly Gly Ser Gln Val 370 375 380 Lys Leu Glu Glu Ser Gly Gly Gly Ser Val Gln Thr Gly Gly Ser Leu 385 390 395 400 Arg Leu Thr Cys Ala Ala Ser Gly Arg Thr Ser Arg Ser Tyr Gly Met 405 410 415 Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ser Gly 420 425 430 Ile Ser Trp Arg Gly Asp Ser Thr Gly Tyr Ala Asp Ser Val Lys Gly 435 440 445 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Asp Leu Gln 450 455 460 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Ala Ala 465 470 475 480 Ala Ala Gly Ser Ala Trp Tyr Gly Thr Leu Tyr Glu Tyr Asp Tyr Trp 485 490 495 Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ser Gly Gly Pro Gly Pro 500 505 510 Ala Gly Met Lys Gly Leu Pro Gly Ser Gln Thr Val Val Thr Gln Glu 515 520 525 Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Gly 530 535 540 Ser Ser Thr Gly Ala Val Thr Ser Gly Asn Tyr Pro Asn Trp Val Gln 545 550 555 560 Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Asp Tyr Lys Asp 565 570 575 Asp Asp Asp Lys Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly 580 585 590 Gly Lys Ala Ala Leu Thr Leu Ser Gly Val Gln Pro Glu Asp Glu Ala 595 600 605 Glu Tyr Tyr Cys Val Leu Trp Tyr Ser Asn Arg Trp Val Phe Gly Gly 610 615 620 Gly Thr Lys Leu Thr Val Leu Gly Gly Gly Ser Gly Gly Gly Ser Glu 625 630 635 640 Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser 645 650 655 Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Tyr Ala 660 665 670 Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala 675 680 685 Arg Ile Arg Ser Lys Tyr Asp Tyr Lys Asp Asp Asp Asp Lys Ala Asp 690 695 700 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 705 710 715 720 Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val Tyr 725 730 735 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Ile Ser Tyr Trp 740 745 750 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly 755 760 765 Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly 770 775 780 Leu Val Gln Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 785 790 795 800 Phe Thr Phe Ser Lys Phe Gly Met Ser Trp Val Arg Gln Ala Pro Gly 805 810 815 Lys Gly Leu Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Arg Asp Thr 820 825 830 Leu Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 835 840 845 Ala Lys Thr Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp 850 855 860 Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Val Ser Ser 865 870 875 880 Gln Gly Thr Leu Val Thr Val Ser Ser 885 <210> 232 <211> 890 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct - Pro140b form 1 <400> 232 Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Ser Val Gln Thr Gly Gly 1 5 10 15 Ser Leu Arg Leu Thr Cys Ala Ala Ser Gly Arg Thr Ser Arg Ser Tyr 20 25 30 Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ser Gly Ile Ser Trp Arg Gly Asp Ser Thr Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Asp 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Ala Ala Ala Gly Ser Ala Trp Tyr Gly Thr Leu Tyr Glu Tyr Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Ser 115 120 125 Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val 130 135 140 Gln Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr 145 150 155 160 Phe Asn Lys Tyr Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly 165 170 175 Leu Glu Trp Val Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr 180 185 190 Tyr Tyr Ala Asp Gln Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp 195 200 205 Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp 210 215 220 Thr Ala Val Tyr Tyr Cys Val Arg His Ala Asn Phe Gly Asn Ser Tyr 225 230 235 240 Ile Ser Tyr Trp Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 245 250 255 Ser Lys Lys Leu Ala Asp Glu Pro Glu Gln Thr Val Val Thr Gln Glu 260 265 270 Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Ala 275 280 285 Ser Ser Thr Gly Ala Val Thr Ser Gly Asn Tyr Pro Asn Trp Val Gln 290 295 300 Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr Lys Phe 305 310 315 320 Leu Val Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly 325 330 335 Lys Ala Ala Leu Thr Leu Ser Gly Val Gln Pro Glu Asp Glu Ala Glu 340 345 350 Tyr Tyr Cys Thr Leu Trp Tyr Ser Asn Arg Trp Val Phe Gly Gly Gly 355 360 365 Thr Lys Leu Thr Val Leu Gly Gly Gly Ser Gly Gly Gly Ser Gln Val 370 375 380 Lys Leu Glu Glu Ser Gly Gly Gly Ser Val Gln Thr Gly Gly Ser Leu 385 390 395 400 Arg Leu Thr Cys Ala Ala Ser Gly Arg Thr Ser Arg Ser Tyr Gly Met 405 410 415 Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ser Gly 420 425 430 Ile Ser Trp Arg Gly Asp Ser Thr Gly Tyr Ala Asp Ser Val Lys Gly 435 440 445 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Asp Leu Gln 450 455 460 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Ala Ala 465 470 475 480 Ala Ala Gly Ser Ala Trp Tyr Gly Thr Leu Tyr Glu Tyr Asp Tyr Trp 485 490 495 Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Ser Lys Lys 500 505 510 Leu Ala Asp Glu Pro Glu Gly Gly Gly Ser Gln Thr Val Val Thr Gln 515 520 525 Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys 530 535 540 Gly Ser Ser Thr Gly Ala Val Thr Ser Gly Asn Tyr Pro Asn Trp Val 545 550 555 560 Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Asp Tyr Lys 565 570 575 Asp Asp Asp Asp Lys Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu 580 585 590 Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val Gln Pro Glu Asp Glu 595 600 605 Ala Glu Tyr Tyr Cys Val Leu Trp Tyr Ser Asn Arg Trp Val Phe Gly 610 615 620 Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Gly Ser Gly Gly Gly Ser 625 630 635 640 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 645 650 655 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Tyr 660 665 670 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 675 680 685 Ala Arg Ile Arg Ser Lys Tyr Asp Tyr Lys Asp Asp Asp Asp Lys Ala 690 695 700 Asp Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn 705 710 715 720 Thr Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val 725 730 735 Tyr Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Ile Ser Tyr 740 745 750 Trp Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly 755 760 765 Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly 770 775 780 Gly Leu Val Gln Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala Ser 785 790 795 800 Gly Phe Thr Phe Ser Lys Phe Gly Met Ser Trp Val Arg Gln Ala Pro 805 810 815 Gly Lys Gly Leu Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Arg Asp 820 825 830 Thr Leu Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp 835 840 845 Asn Ala Lys Thr Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu 850 855 860 Asp Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Val Ser 865 870 875 880 Ser Gln Gly Thr Leu Val Thr Val Ser Ser 885 890 <210> 233 <211> 889 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct - Pro186 form 2 <400> 233 Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Ser Val Gln Thr Gly Gly 1 5 10 15 Ser Leu Arg Leu Thr Cys Ala Ala Ser Gly Arg Thr Ser Arg Ser Tyr 20 25 30 Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ser Gly Ile Ser Trp Arg Gly Asp Ser Thr Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Asp 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Ala Ala Ala Gly Ser Ala Trp Tyr Gly Thr Leu Tyr Glu Tyr Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Ser 115 120 125 Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val 130 135 140 Gln Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr 145 150 155 160 Phe Asn Lys Tyr Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly 165 170 175 Leu Glu Trp Val Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr 180 185 190 Tyr Tyr Ala Asp Gln Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp 195 200 205 Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp 210 215 220 Thr Ala Val Tyr Tyr Cys Val Arg His Ala Asn Phe Gly Asn Ser Tyr 225 230 235 240 Ile Ser Tyr Trp Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 245 250 255 Ser Gly Gly Gly Ser Gly Gly Gly Ser Gln Thr Val Val Thr Gln Glu 260 265 270 Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Ala 275 280 285 Ser Ser Thr Gly Ala Val Thr Ser Gly Asn Tyr Pro Asn Trp Val Gln 290 295 300 Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr Lys Phe 305 310 315 320 Leu Val Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly 325 330 335 Lys Ala Ala Leu Thr Leu Ser Gly Val Gln Pro Glu Asp Glu Ala Glu 340 345 350 Tyr Tyr Cys Thr Leu Trp Tyr Ser Asn Arg Trp Val Phe Gly Gly Gly 355 360 365 Thr Lys Leu Thr Val Leu Gly Gly Gly Ser Gly Gly Gly Ser Gln Val 370 375 380 Lys Leu Glu Glu Ser Gly Gly Gly Ser Val Gln Thr Gly Gly Ser Leu 385 390 395 400 Arg Leu Thr Cys Ala Ala Ser Gly Arg Thr Ser Arg Ser Tyr Gly Met 405 410 415 Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ser Gly 420 425 430 Ile Ser Trp Arg Gly Asp Ser Thr Gly Tyr Ala Asp Ser Val Lys Gly 435 440 445 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Asp Leu Gln 450 455 460 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Ala Ala 465 470 475 480 Ala Ala Gly Ser Ala Trp Tyr Gly Thr Leu Tyr Glu Tyr Asp Tyr Trp 485 490 495 Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ser Gly Gly Pro Gly Pro 500 505 510 Ala Gly Met Lys Gly Leu Pro Gly Ser Gln Thr Val Val Thr Gln Glu 515 520 525 Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Gly 530 535 540 Ser Ser Thr Gly Ala Val Thr Ser Gly Asn Tyr Pro Asn Trp Val Gln 545 550 555 560 Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Asp Tyr Lys Asp 565 570 575 Asp Asp Asp Lys Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly 580 585 590 Gly Lys Ala Ala Leu Thr Leu Ser Gly Val Gln Pro Glu Asp Glu Ala 595 600 605 Glu Tyr Tyr Cys Val Leu Trp Tyr Ser Asn Arg Trp Val Phe Gly Gly 610 615 620 Gly Thr Lys Leu Thr Val Leu Gly Gly Gly Ser Gly Gly Gly Ser Glu 625 630 635 640 Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser 645 650 655 Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Tyr Ala 660 665 670 Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala 675 680 685 Arg Ile Arg Ser Lys Tyr Asp Tyr Lys Asp Asp Asp Asp Lys Ala Asp 690 695 700 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 705 710 715 720 Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val Tyr 725 730 735 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Ile Ser Tyr Trp 740 745 750 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly 755 760 765 Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly 770 775 780 Leu Val Gln Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 785 790 795 800 Phe Thr Phe Ser Lys Phe Gly Met Ser Trp Val Arg Gln Ala Pro Gly 805 810 815 Lys Gly Leu Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Arg Asp Thr 820 825 830 Leu Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 835 840 845 Ala Lys Thr Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp 850 855 860 Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Val Ser Ser 865 870 875 880 Gln Gly Thr Leu Val Thr Val Ser Ser 885 <210> 234 <211> 890 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct - Pro187 form 2 <400> 234 Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Ser Val Gln Thr Gly Gly 1 5 10 15 Ser Leu Arg Leu Thr Cys Ala Ala Ser Gly Arg Thr Ser Arg Ser Tyr 20 25 30 Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ser Gly Ile Ser Trp Arg Gly Asp Ser Thr Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Asp 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Ala Ala Ala Gly Ser Ala Trp Tyr Gly Thr Leu Tyr Glu Tyr Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Ser 115 120 125 Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val 130 135 140 Gln Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr 145 150 155 160 Phe Asn Lys Tyr Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly 165 170 175 Leu Glu Trp Val Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr 180 185 190 Tyr Tyr Ala Asp Gln Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp 195 200 205 Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp 210 215 220 Thr Ala Val Tyr Tyr Cys Val Arg His Ala Asn Phe Gly Asn Ser Tyr 225 230 235 240 Ile Ser Tyr Trp Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 245 250 255 Ser Gly Gly Gly Ser Gly Gly Gly Ser Gln Thr Val Val Thr Gln Glu 260 265 270 Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Ala 275 280 285 Ser Ser Thr Gly Ala Val Thr Ser Gly Asn Tyr Pro Asn Trp Val Gln 290 295 300 Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr Lys Phe 305 310 315 320 Leu Val Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly 325 330 335 Lys Ala Ala Leu Thr Leu Ser Gly Val Gln Pro Glu Asp Glu Ala Glu 340 345 350 Tyr Tyr Cys Thr Leu Trp Tyr Ser Asn Arg Trp Val Phe Gly Gly Gly 355 360 365 Thr Lys Leu Thr Val Leu Gly Gly Gly Ser Gly Gly Gly Ser Gln Val 370 375 380 Lys Leu Glu Glu Ser Gly Gly Gly Ser Val Gln Thr Gly Gly Ser Leu 385 390 395 400 Arg Leu Thr Cys Ala Ala Ser Gly Arg Thr Ser Arg Ser Tyr Gly Met 405 410 415 Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ser Gly 420 425 430 Ile Ser Trp Arg Gly Asp Ser Thr Gly Tyr Ala Asp Ser Val Lys Gly 435 440 445 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Asp Leu Gln 450 455 460 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Ala Ala 465 470 475 480 Ala Ala Gly Ser Ala Trp Tyr Gly Thr Leu Tyr Glu Tyr Asp Tyr Trp 485 490 495 Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Ser Lys Lys 500 505 510 Leu Ala Asp Glu Pro Glu Gly Gly Gly Ser Gln Thr Val Val Thr Gln 515 520 525 Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys 530 535 540 Gly Ser Ser Thr Gly Ala Val Thr Ser Gly Asn Tyr Pro Asn Trp Val 545 550 555 560 Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Asp Tyr Lys 565 570 575 Asp Asp Asp Asp Lys Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu 580 585 590 Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val Gln Pro Glu Asp Glu 595 600 605 Ala Glu Tyr Tyr Cys Val Leu Trp Tyr Ser Asn Arg Trp Val Phe Gly 610 615 620 Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Gly Ser Gly Gly Gly Ser 625 630 635 640 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 645 650 655 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Tyr 660 665 670 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 675 680 685 Ala Arg Ile Arg Ser Lys Tyr Asp Tyr Lys Asp Asp Asp Asp Lys Ala 690 695 700 Asp Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn 705 710 715 720 Thr Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val 725 730 735 Tyr Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Ile Ser Tyr 740 745 750 Trp Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly 755 760 765 Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly 770 775 780 Gly Leu Val Gln Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala Ser 785 790 795 800 Gly Phe Thr Phe Ser Lys Phe Gly Met Ser Trp Val Arg Gln Ala Pro 805 810 815 Gly Lys Gly Leu Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Arg Asp 820 825 830 Thr Leu Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp 835 840 845 Asn Ala Lys Thr Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu 850 855 860 Asp Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Val Ser 865 870 875 880 Ser Gln Gly Thr Leu Val Thr Val Ser Ser 885 890 <210> 235 <211> 895 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct - Pro225 (FL aB7H3 hF7 MMP9 linker) form 2 <400> 235 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Pro Ser Arg Arg Thr Phe His Thr Tyr 20 25 30 His Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Val Ile Asn Trp Ser Gly Gly Ser Thr Val Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Gly Gly Ala Thr Thr Gln Arg Ala Thr Glu Ala Ser Tyr Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Ser 115 120 125 Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val 130 135 140 Gln Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr 145 150 155 160 Phe Asn Lys Tyr Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly 165 170 175 Leu Glu Trp Val Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr 180 185 190 Tyr Tyr Ala Asp Gln Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp 195 200 205 Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp 210 215 220 Thr Ala Val Tyr Tyr Cys Val Arg His Ala Asn Phe Gly Asn Ser Tyr 225 230 235 240 Ile Ser Tyr Trp Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 245 250 255 Ser Gly Gly Gly Ser Gly Gly Gly Ser Gln Thr Val Val Thr Gln Glu 260 265 270 Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Ala 275 280 285 Ser Ser Thr Gly Ala Val Thr Ser Gly Asn Tyr Pro Asn Trp Val Gln 290 295 300 Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr Lys Phe 305 310 315 320 Leu Val Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly 325 330 335 Lys Ala Ala Leu Thr Leu Ser Gly Val Gln Pro Glu Asp Glu Ala Glu 340 345 350 Tyr Tyr Cys Thr Leu Trp Tyr Ser Asn Arg Trp Val Phe Gly Gly Gly 355 360 365 Thr Lys Leu Thr Val Leu Gly Gly Gly Ser Gly Gly Gly Ser Gln Val 370 375 380 Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 385 390 395 400 Arg Leu Ser Cys Ala Pro Ser Arg Arg Thr Phe His Thr Tyr His Met 405 410 415 Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Val 420 425 430 Ile Asn Trp Ser Gly Gly Ser Thr Val Tyr Ala Asp Ser Val Lys Gly 435 440 445 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 450 455 460 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala 465 470 475 480 Gly Gly Ala Thr Thr Gln Arg Ala Thr Glu Ala Ser Tyr Asp Tyr Trp 485 490 495 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ser Gly Gly Pro Gly Pro 500 505 510 Ala Gly Met Lys Gly Leu Pro Gly Ser Gln Thr Val Val Thr Gln Glu 515 520 525 Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Gly 530 535 540 Ser Ser Thr Gly Ala Val Thr Ser Gly Asn Tyr Pro Asn Trp Val Gln 545 550 555 560 Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Asp Tyr Lys Asp 565 570 575 Asp Asp Asp Lys Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly 580 585 590 Gly Lys Ala Ala Leu Thr Leu Ser Gly Val Gln Pro Glu Asp Glu Ala 595 600 605 Glu Tyr Tyr Cys Val Leu Trp Tyr Ser Asn Arg Trp Val Phe Gly Gly 610 615 620 Gly Thr Lys Leu Thr Val Leu Gly Gly Gly Ser Gly Gly Gly Ser Glu 625 630 635 640 Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser 645 650 655 Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Tyr Ala 660 665 670 Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala 675 680 685 Arg Ile Arg Ser Lys Tyr Asp Tyr Lys Asp Asp Asp Asp Lys Ala Asp 690 695 700 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 705 710 715 720 Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val Tyr 725 730 735 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Ile Ser Tyr Trp 740 745 750 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly 755 760 765 Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly 770 775 780 Leu Val Gln Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 785 790 795 800 Phe Thr Phe Ser Lys Phe Gly Met Ser Trp Val Arg Gln Ala Pro Gly 805 810 815 Lys Gly Leu Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Arg Asp Thr 820 825 830 Leu Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 835 840 845 Ala Lys Thr Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp 850 855 860 Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Val Ser Ser 865 870 875 880 Gln Gly Thr Leu Val Thr Val Ser Ser His His His His His His 885 890 895 <210> 236 <211> 891 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct - Pro226 (FL aB7H3 hF12 MMP9 linker) form 2 <400> 236 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ala Ser Pro Arg Thr Phe Ser Thr Tyr 20 25 30 Ser Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Ser Phe Val 35 40 45 Ala Ala Ile Asn Trp Ser Gly Gly Asn Thr Ser Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Gly Gly Val Leu Ala His His Asn Tyr Glu Tyr Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Ser Gly Gly 115 120 125 Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 130 135 140 Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn 145 150 155 160 Lys Tyr Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 165 170 175 Trp Val Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr 180 185 190 Ala Asp Gln Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys 195 200 205 Asn Thr Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala 210 215 220 Val Tyr Tyr Cys Val Arg His Ala Asn Phe Gly Asn Ser Tyr Ile Ser 225 230 235 240 Tyr Trp Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly 245 250 255 Gly Gly Ser Gly Gly Gly Ser Gln Thr Val Val Thr Gln Glu Pro Ser 260 265 270 Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Ala Ser Ser 275 280 285 Thr Gly Ala Val Thr Ser Gly Asn Tyr Pro Asn Trp Val Gln Gln Lys 290 295 300 Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr Lys Phe Leu Val 305 310 315 320 Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly Lys Ala 325 330 335 Ala Leu Thr Leu Ser Gly Val Gln Pro Glu Asp Glu Ala Glu Tyr Tyr 340 345 350 Cys Thr Leu Trp Tyr Ser Asn Arg Trp Val Phe Gly Gly Gly Thr Lys 355 360 365 Leu Thr Val Leu Gly Gly Gly Ser Gly Gly Gly Ser Gln Val Gln Leu 370 375 380 Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu 385 390 395 400 Ser Cys Glu Ala Ser Pro Arg Thr Phe Ser Thr Tyr Ser Met Ala Trp 405 410 415 Phe Arg Gln Ala Pro Gly Lys Glu Arg Ser Phe Val Ala Ala Ile Asn 420 425 430 Trp Ser Gly Gly Asn Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe 435 440 445 Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn 450 455 460 Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Gly Gly 465 470 475 480 Val Leu Ala His His Asn Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly Thr 485 490 495 Leu Val Thr Val Ser Ser Ser Gly Gly Pro Gly Pro Ala Gly Met Lys 500 505 510 Gly Leu Pro Gly Ser Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr 515 520 525 Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly 530 535 540 Ala Val Thr Ser Gly Asn Tyr Pro Asn Trp Val Gln Gln Lys Pro Gly 545 550 555 560 Gln Ala Pro Arg Gly Leu Ile Gly Asp Tyr Lys Asp Asp Asp Asp Lys 565 570 575 Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala 580 585 590 Leu Thr Leu Ser Gly Val Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys 595 600 605 Val Leu Trp Tyr Ser Asn Arg Trp Val Phe Gly Gly Gly Thr Lys Leu 610 615 620 Thr Val Leu Gly Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Val 625 630 635 640 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Lys Leu Ser 645 650 655 Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Tyr Ala Met Asn Trp Val 660 665 670 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Arg Ser 675 680 685 Lys Tyr Asp Tyr Lys Asp Asp Asp Asp Lys Ala Asp Ser Val Lys Asp 690 695 700 Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Ala Tyr Leu Gln 705 710 715 720 Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg 725 730 735 His Gly Asn Phe Gly Asn Ser Tyr Ile Ser Tyr Trp Ala Tyr Trp Gly 740 745 750 Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 755 760 765 Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 770 775 780 Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 785 790 795 800 Lys Phe Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 805 810 815 Trp Val Ser Ser Ile Ser Gly Ser Gly Arg Asp Thr Leu Tyr Ala Glu 820 825 830 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr 835 840 845 Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr 850 855 860 Tyr Cys Thr Ile Gly Gly Ser Leu Ser Val Ser Ser Gln Gly Thr Leu 865 870 875 880 Val Thr Val Ser Ser His His His His His His 885 890 <210> 237 <211> 895 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct - Pro233 (humanized Pro186) form 2 <400> 237 Gln Val Lys Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Gly 1 5 10 15 Ser Leu Thr Leu Ser Cys Ala Ala Ser Gly Arg Thr Ser Arg Ser Tyr 20 25 30 Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ser Gly Ile Ser Trp Arg Gly Asp Ser Thr Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Ala Ala Ala Gly Ser Ala Trp Tyr Gly Thr Leu Tyr Glu Tyr Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Ser 115 120 125 Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val 130 135 140 Gln Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr 145 150 155 160 Phe Asn Lys Tyr Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly 165 170 175 Leu Glu Trp Val Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr 180 185 190 Tyr Tyr Ala Asp Gln Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp 195 200 205 Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp 210 215 220 Thr Ala Val Tyr Tyr Cys Val Arg His Ala Asn Phe Gly Asn Ser Tyr 225 230 235 240 Ile Ser Tyr Trp Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 245 250 255 Ser Gly Gly Gly Ser Gly Gly Gly Ser Gln Thr Val Val Thr Gln Glu 260 265 270 Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Ala 275 280 285 Ser Ser Thr Gly Ala Val Thr Ser Gly Asn Tyr Pro Asn Trp Val Gln 290 295 300 Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr Lys Phe 305 310 315 320 Leu Val Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly 325 330 335 Lys Ala Ala Leu Thr Leu Ser Gly Val Gln Pro Glu Asp Glu Ala Glu 340 345 350 Tyr Tyr Cys Thr Leu Trp Tyr Ser Asn Arg Trp Val Phe Gly Gly Gly 355 360 365 Thr Lys Leu Thr Val Leu Gly Gly Gly Ser Gly Gly Gly Ser Gln Val 370 375 380 Lys Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Gly Ser Leu 385 390 395 400 Thr Leu Ser Cys Ala Ala Ser Gly Arg Thr Ser Arg Ser Tyr Gly Met 405 410 415 Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ser Gly 420 425 430 Ile Ser Trp Arg Gly Asp Ser Thr Gly Tyr Ala Asp Ser Val Lys Gly 435 440 445 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln 450 455 460 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Ala 465 470 475 480 Ala Ala Gly Ser Ala Trp Tyr Gly Thr Leu Tyr Glu Tyr Asp Tyr Trp 485 490 495 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ser Gly Gly Pro Gly Pro 500 505 510 Ala Gly Met Lys Gly Leu Pro Gly Ser Gln Thr Val Val Thr Gln Glu 515 520 525 Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Gly 530 535 540 Ser Ser Thr Gly Ala Val Thr Ser Gly Asn Tyr Pro Asn Trp Val Gln 545 550 555 560 Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Asp Tyr Lys Asp 565 570 575 Asp Asp Asp Lys Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly 580 585 590 Gly Lys Ala Ala Leu Thr Leu Ser Gly Val Gln Pro Glu Asp Glu Ala 595 600 605 Glu Tyr Tyr Cys Val Leu Trp Tyr Ser Asn Arg Trp Val Phe Gly Gly 610 615 620 Gly Thr Lys Leu Thr Val Leu Gly Gly Gly Ser Gly Gly Gly Ser Glu 625 630 635 640 Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser 645 650 655 Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Tyr Ala 660 665 670 Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala 675 680 685 Arg Ile Arg Ser Lys Tyr Asp Tyr Lys Asp Asp Asp Asp Lys Ala Asp 690 695 700 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 705 710 715 720 Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val Tyr 725 730 735 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Ile Ser Tyr Trp 740 745 750 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly 755 760 765 Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly 770 775 780 Leu Val Gln Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 785 790 795 800 Phe Thr Phe Ser Lys Phe Gly Met Ser Trp Val Arg Gln Ala Pro Gly 805 810 815 Lys Gly Leu Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Arg Asp Thr 820 825 830 Leu Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 835 840 845 Ala Lys Thr Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp 850 855 860 Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Val Ser Ser 865 870 875 880 Gln Gly Thr Leu Val Thr Val Ser Ser His His His His His His 885 890 895 <210> 238 <211> 875 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct - Pro311 (FL aFOLR1 h77.2 MMP9 linker) form 2 <400> 238 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Asn Ser 20 25 30 Val Met Ala Trp Tyr Arg Gln Thr Pro Gly Asn Glu Arg Glu Phe Val 35 40 45 Ala Ile Ile Asn Ser Ile Gly Ile Thr Asn Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Val Cys Asn 85 90 95 Arg Asn Phe Asp Arg Ile Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser Gly Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu 115 120 125 Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Lys Leu Ser Cys 130 135 140 Ala Ala Ser Gly Phe Thr Phe Asn Lys Tyr Ala Ile Asn Trp Val Arg 145 150 155 160 Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Arg Ser Lys 165 170 175 Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Gln Val Lys Asp Arg Phe 180 185 190 Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn 195 200 205 Asn Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg His Ala 210 215 220 Asn Phe Gly Asn Ser Tyr Ile Ser Tyr Trp Ala Tyr Trp Gly Gln Gly 225 230 235 240 Thr Leu Val Thr Val Ser Ser Gly Gly Gly Ser Gly Gly Gly Ser Gln 245 250 255 Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr 260 265 270 Val Thr Leu Thr Cys Ala Ser Ser Thr Gly Ala Val Thr Ser Gly Asn 275 280 285 Tyr Pro Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu 290 295 300 Ile Gly Gly Thr Lys Phe Leu Val Pro Gly Thr Pro Ala Arg Phe Ser 305 310 315 320 Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val Gln 325 330 335 Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Thr Leu Trp Tyr Ser Asn Arg 340 345 350 Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Gly Ser 355 360 365 Gly Gly Gly Ser Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val 370 375 380 Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr 385 390 395 400 Val Ser Asn Ser Val Met Ala Trp Tyr Arg Gln Thr Pro Gly Asn Glu 405 410 415 Arg Glu Phe Val Ala Ile Ile Asn Ser Ile Gly Ile Thr Asn Tyr Ala 420 425 430 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn 435 440 445 Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 450 455 460 Tyr Val Cys Asn Arg Asn Phe Asp Arg Ile Tyr Trp Gly Gln Gly Thr 465 470 475 480 Leu Val Thr Val Ser Ser Ser Gly Gly Pro Gly Pro Ala Gly Met Lys 485 490 495 Gly Leu Pro Gly Ser Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr 500 505 510 Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly 515 520 525 Ala Val Thr Ser Gly Asn Tyr Pro Asn Trp Val Gln Gln Lys Pro Gly 530 535 540 Gln Ala Pro Arg Gly Leu Ile Gly Asp Tyr Lys Asp Asp Asp Asp Lys 545 550 555 560 Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala 565 570 575 Leu Thr Leu Ser Gly Val Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys 580 585 590 Val Leu Trp Tyr Ser Asn Arg Trp Val Phe Gly Gly Gly Thr Lys Leu 595 600 605 Thr Val Leu Gly Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Val 610 615 620 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Lys Leu Ser 625 630 635 640 Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Tyr Ala Met Asn Trp Val 645 650 655 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Arg Ser 660 665 670 Lys Tyr Asp Tyr Lys Asp Asp Asp Asp Lys Ala Asp Ser Val Lys Asp 675 680 685 Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Ala Tyr Leu Gln 690 695 700 Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg 705 710 715 720 His Gly Asn Phe Gly Asn Ser Tyr Ile Ser Tyr Trp Ala Tyr Trp Gly 725 730 735 Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 740 745 750 Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 755 760 765 Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 770 775 780 Lys Phe Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 785 790 795 800 Trp Val Ser Ser Ile Ser Gly Ser Gly Arg Asp Thr Leu Tyr Ala Glu 805 810 815 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr 820 825 830 Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr 835 840 845 Tyr Cys Thr Ile Gly Gly Ser Leu Ser Val Ser Ser Gln Gly Thr Leu 850 855 860 Val Thr Val Ser Ser His His His His His His 865 870 875 <210> 239 <211> 873 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct - Pro312 (FL aFOLR1 h59.3 MMP9 linker) <400> 239 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Pro Gly Asn Thr Phe Ser Ile Ser 20 25 30 Ala Met Gly Trp ...

Claims

1. A single domain antigen binding domain that binds to human Trop2, wherein the single domain antigen binding domain comprises: (i) a complementarity determining region 1 consisting of the amino acid sequence of SEQ ID NO: 78, a complementarity determining region 2 consisting of the amino acid sequence of SEQ ID NO: 79, and a complementarity determining region 3 consisting of the amino acid sequence of SEQ ID NO: 80; or (ii) complementarity determining region 1 consisting of the amino acid sequence of SEQ ID NO:94, complementarity determining region 2 consisting of the amino acid sequence of SEQ ID NO:95, and complementarity determining region 3 consisting of the amino acid sequence of SEQ ID NO:

96.

2. A single domain antigen binding domain according to claim 1, wherein the single domain antigen binding domain comprises a complementarity determining region 1 consisting of the amino acid sequence of SEQ ID NO: 78, a complementarity determining region 2 consisting of the amino acid sequence of SEQ ID NO: 79, and a complementarity determining region 3 consisting of the amino acid sequence of SEQ ID NO:

80.

3. The single domain antigen binding domain of claim 2, wherein the single domain antigen binding domain comprises the amino acid sequence of SEQ ID NO:

77.

4. A single domain antigen binding domain according to claim 1, wherein the single domain antigen binding domain comprises a complementarity determining region 1 consisting of the amino acid sequence of SEQ ID NO: 94, a complementarity determining region 2 consisting of the amino acid sequence of SEQ ID NO: 95, and a complementarity determining region 3 consisting of the amino acid sequence of SEQ ID NO:

96.

5. The single domain antigen binding domain of claim 4, wherein the single domain antigen binding domain comprises the amino acid sequence of SEQ ID NO:

93.

6. A nucleic acid encoding the single domain antigen binding domain according to any one of claims 1 to 5.

7. An expression vector comprising the nucleic acid according to claim 6. A host cell comprising the expression vector according to claim 7 .

9. A method for preparing a single domain antigen binding domain, the method comprising culturing the host cell of claim 8 under conditions wherein the single domain antigen binding domain is expressed and recovering the single domain antigen binding domain.

Images