RU2804458C2 - 5t4 and 4-1bb antigen-binding proteins and related compositions and methods - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: invention relates to a bispecific polypeptide for use in the treatment of a disorder associated with the expression of 5T4. Also a method of treating cancer in a subject with the help of the said antibody is disclosed.

EFFECT: invention makes it possible to effectively treat cancer characterized by 5T4 expression.

20 cl, 40 dwg, 23 ex, 33 tbl

Description

Cross reference to related applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 62/535,107, filed July 20, 2017; 62/575,820 filed Oct. 23, 2017; and 62/648,072, filed March 26, 2018, the contents of each of which are incorporated herein by reference in their entirety.

Description of the text file submitted electronically

[0002] The contents of the text file filed electronically with this application are incorporated herein by reference in their entirety. A copy of the sequence listing in machine-readable format (file name: APVO_057_03WO_SeqList_ST25.txt, recording date: July 20, 2018, file size: 260 kilobytes).

Field of technology to which the invention relates

[0003] The present invention relates to molecules that specifically bind to trophoblastic glycoprotein (5T4). These molecules may have at least one humanized 5T4 binding domain. These molecules are useful for characterizing or treating disorders characterized by 5T4 expression, such as cancer. The protein therapeutic agent that binds to 5T4 may be a monospecific protein therapeutic agent or a multispecific protein therapeutic agent. The present invention also provides protein therapeutics that specifically bind to tumor necrosis factor receptor superfamily member 9 (4-1BB or CD137). A multispecific protein therapeutic can bind both 5T4-expressing cells and 4-1BB expressed on effector cells to enhance activation, effector cell proliferation, and/or effector cell-mediated cytotoxicity.

State of the art

[0004] 5T4 (also called trophoblastic glycoprotein, TPBG, M6P1 and Waif1) is a well-characterized tumor-associated antigen (TAA), first identified by Professor Peter Stern of the University of Manchester (Hole and Stern, 1988). It is an oncofetal antigen expressed in a significant proportion of patients with a variety of malignancies, including non-small cell lung cancer, renal, pancreatic, prostate, breast, colorectal, gastric, ovarian and cervical cancers, as well as acute lymphocytic leukemia , and has also been shown to be expressed in tumor-initiating cells (Castro et al. , 2012; Damelin et al. , 2011; Elkord et al. , 2009; Southall et al. , 1990).

[0005] Although low levels of 5T4 expression have been detected in some healthy tissues, such as the placenta and specialized epithelium, expression levels in tumors are significantly higher.

[0006] Evidence suggests that 5T4 regulates the functional activity of CXCR4 (Castro et al. , 2012; Southgate et al. , 2010). 5T4-binding antibodies or 5T4 knockdown resulted in inhibition of CXCR4-mediated cell migration, a pathway involved in tumor growth and metastasis. Therefore, targeting 5T4 may provide therapeutic benefits in the treatment of various cancers. There are currently no FDA-approved therapeutics that specifically target or bind 5T4. There is a need for new therapeutic agents for the treatment of malignancies characterized by 5T4 expression.

[0007] 4-1BB (also known as CD137 or TNFRSF9) is a member of the tumor necrosis factor (TNF) receptor (TNFR) superfamily. 4-1BB is expressed on cells from a variety of cell populations, including activated CD4 ⁺ and CD8 ⁺ T cells, regulatory T cells (Tregs), dendritic cells (DCs), monocytes, mast cells, eosinophils, and tumor endothelial cells. Activation of 4-1BB is dependent on receptor oligomerization (Rabu et al., 2005; Wyzgol et al., 2009) caused by binding to 4-1BBL (also known as CD137L), which is expressed as a trimer on the cell surface of antigen-presenting cells (APCs). and other cell types. Activation of 4-1BB on CD8 ⁺ T cells promotes and enhances CD8 ⁺ T cell effector functions, and selectively promotes the production of Th1 cytokines (Shuford et al. , 1997; Lee et al ., 2002; Pulle et al ., 2006) . Activation of 4-1BB on CD4 ⁺ T cells, 4-1BB stimulation first leads to activation and then to activation-induced cell death, which is believed to explain why 4-1BB agonist antibodies have shown therapeutic effects on tumor immunity , as well as autoimmunity (Zhang, JCI, 2007; Sun, Trends Mol Med, 2003). 4-1BB activation has also been reported to suppress Tregs function or convert Tregs into cytotoxic CD4 ⁺ T cells (Akhmetzyanova et al. , 2016; So et al ., 2008).

[0008] In addition to expression on T cells and modulation of their effector function, 4-1BB expression is increased on CD16- and cytokine-activated natural killer (NK) cells. Activation of 4-1BB has been shown to enhance antibody-dependent cellular cytotoxicity (ADCC) activity of NK cells in both murine and human cells (Kohrt 2012 and 2014 J Clin Invest, reviewed by Hout 2012, Oncoimm). In addition, activation of 4-1BB expressed on APCs such as DCs and macrophages can also induce and/or modulate immune activation.

[0009] The role of 4-1BB in modulating immune cell activation suggests that it may be a suitable target for immunotherapy in the treatment of multiple types of cancer. Indeed, two antibodies to 4-1BB are in clinical trials: urelumab (BMS-66513), developed by Bristol-Myers Squibb, and PF-05082566, developed by Pfizer. Phase I and II studies are ongoing in various indications for each antibody. However, when 4-1BB is activated, liver and skin toxicity has been observed in patients and mouse models (Ascierto et al ., 2010; Dubrot et al ., 2010; Niu et al ., 2007). Additionally, a phase II study of urelumab as second-line therapy for metastatic melanoma was stopped in 2009 due to liver toxicity (Garber et al ., 2011; Li and Liu, 2013).

[0010] Therefore, there remains a need in the art for therapeutic agents that safely and effectively activate 4-1BB for use in the treatment of cancer.

Brief description of the invention

[0011] In some aspects, the present invention provides polyspecific polypeptides that specifically bind to 5T4 and/or 4-1BB. In some embodiments, the 5T4 binding domain of the invention binds to the extracellular domain of human 5T4. In certain embodiments, the 4-1BB binding domain of the invention binds to the extracellular domain of human 4-1BB. In some embodiments, the invention provides a polyspecific polypeptide comprising a 5T4 binding domain that specifically binds to human 5T4 and a 4-1BB binding domain, wherein said 5T4 binding domain comprises: (i) an immunoglobulin heavy chain variable region containing HCDR1, HCDR2 and HCDR3, and (ii) an immunoglobulin light chain variable region comprising LCDR1, LCDR2 and LCDR3, wherein (a) HCDR1 contains the amino acid sequence of SEQ ID NO: 30; (b) HCDR2 contains the amino acid sequence of SEQ ID NO: 32; (c) HCDR3 contains the amino acid sequence of SEQ ID NO: 34; (d) LCDR1 contains the amino acid sequence of SEQ ID NO: 42; (e) LCDR2 contains the amino acid sequence of SEQ ID NO: 10; and (f) LCDR3 comprises the amino acid sequence of SEQ ID NO: 36. In some embodiments, the 5T4 binding domain comprising the CDR sequences set forth above comprises a heavy chain variable region comprising an amino acid sequence having at least 90% 95% or at least 97% identity to a sequence selected from the group consisting of SEQ ID NOs: 38 and 46. In certain embodiments, the 5T4 binding domain comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 38 and 46. In other embodiments, the 5T4 binding domain comprising the CDR sequences set forth above comprises a light chain variable region comprising an amino acid sequence having at least 90%, at least 95%, or at least 97% identity to a sequence selected from the group consisting of SEQ ID NOs: 44, 48, and 50. In some embodiments, the 5T4 binding domain comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 44, 48, and 50. In other embodiments, the 5T4 binding domain comprising the CDR sequences set forth above comprises i) a heavy chain variable region comprising an amino acid sequence having at least 90%, at least 95%, or at least 97% identity to SEQ ID NO: 38, and a light chain variable region comprising an amino acid sequence having at least 90%, at least 95%, or at least 97% identity to SEQ ID NO: 44; or ii) a heavy chain variable region comprising an amino acid sequence having at least 90%, at least 95% or at least 97% identity to SEQ ID NO: 46, and a light chain variable region comprising an amino acid sequence having at least at least 90%, at least 95%, or at least 97% identical to SEQ ID NO: 48; or iii) a heavy chain variable region comprising an amino acid sequence having at least 90%, at least 95% or at least 97% identity to SEQ ID NO: 46, and a light chain variable region comprising an amino acid sequence having at least at least 90%, at least 95%, or at least 97% identical to SEQ ID NO: 5. In some embodiments, the 5T4 binding domain comprises i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 38, and a variable region light chain containing the amino acid sequence SEQ ID NO: 44; or ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 46 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 48; or iii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 46 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 50.

[0012] In certain aspects, the invention includes a polyspecific polypeptide comprising a 5T4 binding domain that specifically binds to human 5T4 and a 4-1BB binding domain that specifically binds to human 4-1BB, wherein each of the 4-1BB binding domain and The 5T4 binding domain contains (i) an immunoglobulin heavy chain variable region (VH) containing HCDR1, HCDR2 and HCDR3; and (ii) an immunoglobulin light chain (VL) variable region comprising LCDR1, LCDR2 and LCDR3, wherein the VH and/or VL region of the 5T4 binding domain contains one or more mutations in the framework region; and wherein the 5T4 binding domain comprises (a) HCDR1 comprising the amino acid sequence of SEQ ID NO: 30; (b) HCDR2 containing the amino acid sequence of SEQ ID NO: 32; (c) HCDR3 containing the amino acid sequence of SEQ ID NO: 34; (d) LCDR1 containing the amino acid sequence of SEQ ID NO: 8; (e) LCDR2 containing the amino acid sequence of SEQ ID NO: 10; and (f) LCDR3 comprising the amino acid sequence of SEQ ID NO: 36. In some embodiments, the 5T4 binding domain comprising the CDR sequences set forth above comprises a heavy chain variable region comprising an amino acid sequence having at least 90% at least 95% or at least 97% identical to SEQ ID NO: 38. In one embodiment, the 5T4 binding domain comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 38. In some embodiments, the 5T4 binding domain comprising CDR sequences set forth above comprises a light chain variable region comprising an amino acid sequence having at least 90%, at least 95%, or at least 97% identity to SEQ ID NO: 40. In certain embodiments, the 5T4 binding domain comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 40. In some embodiments, the 5T4 binding domain comprising the CDR sequences set forth above comprises a heavy chain variable region comprising an amino acid sequence having at least 90% 95% or at least 97% identity to SEQ ID NO: 38, and a light chain variable region comprising an amino acid sequence having at least 90%, at least 95% or at least 97% identity to SEQ ID NO: 40. In some embodiments, the 5T4 binding domain comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 38 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 40.

[0013] The invention also provides a polyspecific polypeptide comprising a 5T4 binding domain that specifically binds to human 5T4 and a 4-1BB binding domain that specifically binds to human 4-1BB, wherein each of the 4-1BB binding domain and 5T4 The α-binding domain contains (i) an immunoglobulin heavy chain variable region (VH) containing HCDR1, HCDR2 and HCDR3; and (ii) an immunoglobulin light chain (VL) variable region comprising LCDR1, LCDR2 and LCDR3, wherein the VH and/or VL region of the 4-1BB binding domain contains one or more mutations in the framework region; and wherein the 5T4 binding domain comprises (a) HCDR1 comprising the amino acid sequence of SEQ ID NO: 30; (b) HCDR2 containing the amino acid sequence of SEQ ID NO: 32; c) HCDR3 containing the amino acid sequence SEQ ID NO: 34; (d) LCDR1 containing the amino acid sequence of SEQ ID NO: 42; (e) LCDR2 containing the amino acid sequence of SEQ ID NO: 10; and (f) LCDR3 comprising the amino acid sequence of SEQ ID NO: 36. In some embodiments, the 5T4 binding domain comprising the CDR sequences set forth above comprises a heavy chain variable region comprising an amino acid sequence having at least 90% at least 95% or at least 97% identity to a sequence selected from the group consisting of SEQ ID NOs: 38 and 46. In certain embodiments, the 5T4 binding domain comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of from SEQ ID NOs: 38 and 46. In some embodiments, the 5T4 binding domain comprising the CDR sequences set forth above comprises a light chain variable region comprising an amino acid sequence having at least 90%, at least 95%, or at least at least 97% identity to a sequence selected from the group consisting of SEQ ID NOs: 44, 48 and 50. In some aspects, the 5T4 binding domain comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 44, 48, and 50. In certain embodiments, the 5T4 binding domain comprising the CDR sequences set forth above comprises i) a heavy chain variable region comprising an amino acid sequence having at least 90%, at least 95%, or at least 97% identity to SEQ ID NO: 38, and a light chain variable region comprising an amino acid sequence having at least 90%, at least 95%, or at least 97% identity to SEQ ID NO: 44; or ii) a heavy chain variable region comprising an amino acid sequence having at least 90%, at least 95% or at least 97% identity to SEQ ID NO: 46, and a light chain variable region comprising an amino acid sequence having at least at least 90%, at least 95%, or at least 97% identical to SEQ ID NO: 48; or iii) a heavy chain variable region comprising an amino acid sequence having at least 90%, at least 95% or at least 97% identity to SEQ ID NO: 46, and a light chain variable region comprising an amino acid sequence having at least at least 90%, at least 95%, or at least 97% identical to SEQ ID NO: 50. In some embodiments, the 5T4 binding domain comprises i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 38, and a variable region light chain containing the amino acid sequence SEQ ID NO: 44; or ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 46 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 48; or iii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 46 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 50.

[0014] In some aspects, the invention provides a polyspecific polypeptide comprising a 5T4 binding domain that specifically binds to human 5T4 and a 4-1BB binding domain that specifically binds to human 4-1BB, wherein each of said 4-1BB binding domain and the 5T4 binding domain contains (i) an immunoglobulin heavy chain variable region (VH) containing HCDR1, HCDR2 and HCDR3; and (ii) an immunoglobulin light chain (VL) variable region comprising LCDR1, LCDR2 and LCDR3, wherein the VH and/or VL region of the 4-1BB binding domain contains one or more mutations in the framework region; and wherein the 5T4 binding domain comprises (a) HCDR1 comprising the amino acid sequence of SEQ ID NO: 52; (b) HCDR2 containing the amino acid sequence of SEQ ID NO: 32; (c) HCDR3 containing the amino acid sequence of SEQ ID NO: 34; (d) LCDR1 containing the amino acid sequence of SEQ ID NO: 54; (e) LCDR2 containing the amino acid sequence of SEQ ID NO: 10; and (f) LCDR3 comprising the amino acid sequence of SEQ ID NO: 36. In certain embodiments, the 5T4 binding domain comprising the CDR sequences set forth above comprises a heavy chain variable region comprising an amino acid sequence having at least 90% at least 95% or at least 97% identical to SEQ ID NO: 56. In some aspects, the 5T4 binding domain comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 56. In certain embodiments, the 5T4 binding domain comprising CDR sequences above, comprises a light chain variable region comprising an amino acid sequence having at least 90%, at least 95%, or at least 97% identity to SEQ ID NO: 58. In some embodiments, the 5T4 binding domain comprises a variable region light chain comprising the amino acid sequence of SEQ ID NO: 58. In certain embodiments, the 5T4 binding domain comprising the CDR sequences set forth above comprises a heavy chain variable region comprising an amino acid sequence having at least 90%, at least 95% or at least 97% identity to SEQ ID NO: 56, and a light chain variable region comprising an amino acid sequence having at least 90%, at least 95%, or at least 97% identity to SEQ ID NO: 58. In some In embodiments, the 5T4 binding domain comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 56 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58.

[0015] The invention also provides a polyspecific polypeptide comprising a 5T4 binding domain that specifically binds to human 5T4 and a 4-1BB binding domain that specifically binds to human 4-1BB, wherein each of the 4-1BB binding domain and 5T4 The α-binding domain contains (i) an immunoglobulin heavy chain variable region (VH) containing HCDR1, HCDR2 and HCDR3; and (ii) an immunoglobulin light chain (VL) variable region comprising LCDR1, LCDR2 and LCDR3, wherein the VH and/or VL region of the 4-1BB binding domain contains one or more mutations in the framework region; and wherein the 5T4 binding domain comprises (a) HCDR1 comprising the amino acid sequence of SEQ ID NO: 60; (b) HCDR2 containing the amino acid sequence of SEQ ID NO: 62; (c) HCDR3 containing the amino acid sequence of SEQ ID NO: 34; (d) LCDR1 containing the amino acid sequence of SEQ ID NO: 54; (e) LCDR2 containing the amino acid sequence of SEQ ID NO: 10; and (f) LCDR3 comprising the amino acid sequence of SEQ ID NO: 36. In certain embodiments, the 5T4 binding domain comprising the CDR sequences set forth above comprises a heavy chain variable region comprising an amino acid sequence having at least 90% at least 95% or at least 97% identical to SEQ ID NO: 64. In some embodiments, the 5T4 binding domain comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 64. In certain aspects, the 5T4 binding domain comprising CDR sequences above, comprises a light chain variable region comprising an amino acid sequence having at least 90%, at least 95%, or at least 97% identity to SEQ ID NO: 58. In some embodiments, the 5T4 binding domain comprises a variable region light chain comprising the amino acid sequence of SEQ ID NO: 58. In certain embodiments, the 5T4 binding domain comprising the CDR sequences set forth above comprises a heavy chain variable region comprising an amino acid sequence having at least 90%, at least 95% or at least 97% identity to SEQ ID NO: 64, and a light chain variable region comprising an amino acid sequence having at least 90%, at least 95%, or at least 97% identity to SEQ ID NO: 58. In some In embodiments, the 5T4 binding domain comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 64 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58.

[0016] In certain aspects, the present invention provides a polyspecific polypeptide comprising a 4-1BB binding domain that specifically binds to human 4-1BB. The invention also relates to a polyspecific polypeptide comprising a 4-1BB binding domain and a 5T4 binding domain, wherein said 4-1BB binding domain is linked to said 5T4 binding domain via a binding domain linker. In some embodiments, the polyspecific polypeptide comprises, in an amino-terminal to carboxy-terminal direction, (i) a 5T4 binding domain, (ii) a linker binding domains, and (iii) a 4-1BB binding domain. In certain aspects, the 5T4 binding domain is a single chain variable fragment (scFv). In some embodiments, the light chain variable region of said scFv is carboxy-terminal to the heavy chain variable region of said scFv. In other embodiments, the light chain variable region of said scFv is amino terminal to the heavy chain variable region of said scFv. In certain aspects, the scFv comprises a linker polypeptide. In some examples, the linker polypeptide is located between the light chain variable region and the heavy chain variable region of said scFv. In certain embodiments, the linker polypeptide comprises a Gly ₄ Ser linker. In some embodiments, the linker polypeptide contains a structure of the formula (Gly ₄ Ser) _n , where n = 1-5. In some aspects, the linker polypeptide comprises an amino acid sequence selected from SEQ ID NO: 85-108. In certain embodiments, the linker polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 98 (GGGGSGGGGSGGGGSGGGGS).

[0017] In certain embodiments, a scFv comprising the CDR sequences set forth above comprises a sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 118, 120, 122, 124, 126, 128, 130, 132, 134, and 170. In some embodiments, the scFv comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 118, 120, 122, 124, 126, 128, 130, 132, 134 and 170.

[0018] In some aspects, the invention provides a polyspecific polypeptide comprising a 4-1BB binding domain that specifically binds to human 4-1BB and a 5T4 binding domain, wherein said 4-1BB binding domain comprises: (i) a severe variable region an immunoglobulin chain (VH) containing HCDR1, HCDR2 and HCDR3, and (ii) an immunoglobulin light chain (VL) variable region containing LCDR1, LCDR2 and LCDR3, wherein (a) HCDR1 contains the amino acid sequence of SEQ ID NO: 2; (b) HCDR2 contains the amino acid sequence of SEQ ID NO: 4; (c) HCDR3 contains the amino acid sequence of SEQ ID NO: 6; (d) LCDR1 contains the amino acid sequence of SEQ ID NO: 8; (e) LCDR2 contains the amino acid sequence of SEQ ID NO: 10; and (f) LCDR3 comprises the amino acid sequence of SEQ ID NO: 12. In certain embodiments, the 4-1BB binding domain comprising the CDR sequences set forth above comprises a heavy chain variable region comprising an amino acid sequence having at least 90% at least 95% or at least 97% identical to SEQ ID NO: 14. In some embodiments, the 4-1BB-binding domain comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 14. In certain embodiments, 4-1BB- the binding domain comprising the CDR sequences set forth above comprises a light chain variable region comprising an amino acid sequence having at least 90%, at least 95%, or at least 97% identity to SEQ ID NO: 16. In some embodiments, 4 The -1BB binding domain comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16. In certain embodiments, the 4-1BB binding domain comprising the CDR sequences set forth above comprises a heavy chain variable region comprising the amino acid sequence having at least 90%, at least 95%, or at least 97% identity to SEQ ID NO: 14, and a light chain variable region comprising an amino acid sequence having at least 90%, at least 95%, or at least 97 % identity to SEQ ID NO: 16. In some aspects, the 4-1BB binding domain comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 14 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16.

[0019] The invention also provides a polyspecific polypeptide comprising a 5T4 binding domain specifically binding to human 5T4 and a 4-1BB binding domain, wherein said 4-1BB binding domain comprises (i) an immunoglobulin heavy chain variable region (VH) , containing HCDR1, HCDR2 and HCDR3; and (ii) an immunoglobulin light chain (VL) variable region comprising LCDR1, LCDR2 and LCDR3, and wherein said VH and/or VL region of the 4-1BB binding domain contains one or more mutations in the framework region, and wherein said 4-1BB The β-binding domain contains (a) HCDR1 containing the amino acid sequence of SEQ ID NO: 18; (b) HCDR2 containing the amino acid sequence of SEQ ID NO: 4; (c) HCDR3 containing the amino acid sequence of SEQ ID NO: 6; (d) LCDR1 containing the amino acid sequence of SEQ ID NO: 8; (e) LCDR2 containing the amino acid sequence of SEQ ID NO: 10; and (f) LCDR3 comprising the amino acid sequence of SEQ ID NO: 12. In certain embodiments, the 4-1BB binding domain comprising the CDR sequences set forth above comprises a heavy chain variable region comprising an amino acid sequence having at least 90%, at least 95% or at least 97% identical to SEQ ID NO: 20. In some embodiments, the 4-1BB binding domain comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 20. In certain embodiments, 4-1BB The α-binding domain comprising the CDR sequences set forth above comprises a light chain variable region comprising an amino acid sequence having at least 90%, at least 95%, or at least 97% identity to SEQ ID NO: 22. In some embodiments The 4-1BB binding domain comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 22. In certain embodiments, the 4-1BB binding domain comprising the CDR sequences set forth above comprises a heavy chain variable region comprising the amino acid sequence having at least 90%, at least 95%, or at least 97% identical to SEQ ID NO: 20, and a light chain variable region comprising an amino acid sequence having at least 90%, at least 95%, or at least 97% identity to SEQ ID NO: 22. In some embodiments, the 4-1BB binding domain comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 20 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 22.

[0020] The invention also provides a polyspecific polypeptide comprising a 5T4 binding domain specifically binding to human 5T4 and a 4-1BB binding domain, wherein said 4-1BB binding domain comprises (i) a heavy chain variable region ( _VH ) immunoglobulin containing HCDR1, HCDR2 and HCDR3; and (ii) an immunoglobulin light chain variable region ( _VL ) comprising LCDR1, LCDR2 and LCDR3, and wherein said region of the _VH and/or _VL 4-1BB binding domain contains one or more mutations in the framework region, and wherein said The 4-1BB binding domain contains (a) HCDR1 containing the amino acid sequence of SEQ ID NO: 24; (b) HCDR2 containing the amino acid sequence of SEQ ID NO: 4; (c) HCDR3 containing the amino acid sequence of SEQ ID NO: 6; (d) LCDR1 containing the amino acid sequence of SEQ ID NO: 8; (e) LCDR2 containing the amino acid sequence of SEQ ID NO: 10; and (f) LCDR3 comprising the amino acid sequence of SEQ ID NO: 12. In certain embodiments, the 4-1BB binding domain comprising the CDR sequences set forth above comprises a heavy chain variable region comprising an amino acid sequence having at least 90%, at least 95% or at least 97% identical to SEQ ID NO: 26. In some embodiments, the 4-1BB binding domain comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 26. In certain embodiments, 4-1BB The α-binding domain comprising the CDR sequences set forth above comprises a light chain variable region comprising an amino acid sequence having at least 90%, at least 95%, or at least 97% identity to SEQ ID NO: 16. In some embodiments The 4-1BB binding domain comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16. In certain embodiments, the 4-1BB binding domain comprising the CDR sequences set forth above comprises a heavy chain variable region comprising the amino acid sequence having at least 90%, at least 95%, or at least 97% identical to SEQ ID NO: 26, and a light chain variable region comprising an amino acid sequence having at least 90%, at least 95%, or at least 97% identity to SEQ ID NO: 16. In some embodiments, the 4-1BB binding domain comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 26 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16.

[0021] In certain embodiments, the invention provides a polyspecific polypeptide comprising a 4-1BB binding domain, wherein the 4-1BB binding domain is a single chain variable fragment (scFv). In some aspects, the light chain variable region of said scFv is carboxy-terminal to the heavy chain variable region of said scFv. In other aspects, the light chain variable region of said scFv is amino terminal to the heavy chain variable region of said scFv. In certain aspects, the scFv comprises a linker polypeptide. In some examples, the linker polypeptide is located between the light chain variable region and the heavy chain variable region of said scFv. In certain embodiments, the linker polypeptide comprises a Gly ₄ Ser linker. In some embodiments, the linker polypeptide contains a structure of the formula (Gly ₄ Ser) _n , where n = 1-5. In some aspects, the linker polypeptide comprises an amino acid sequence selected from SEQ ID NO: 85-108. In certain embodiments, the linker polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 98 (GGGGSGGGGSGGGGSGGGGS). In certain aspects, the polyspecific polypeptide of the invention comprises a 4-1BB binding domain linked to a 5T4 binding domain via a binding domain linker. In some embodiments, the polyspecific polypeptide comprises, in an amino-terminal to carboxy-terminal direction, (i) a 5T4 binding domain, (ii) a linker binding domains, and (iii) a 4-1BB binding domain.

[0022] In some embodiments, the 4-1BB binding domain comprising the CDR sequences identified above comprises a sequence having at least 80%, at least 85%, at least 90%, or at least 95% amino acid identity a sequence selected from the group consisting of SEQ ID NOs: 110, 112, 114, and 116. In certain embodiments, the 4-1BB binding domain comprises a sequence selected from the group consisting of SEQ ID NOs: 110, 112, 114, and 116 .

[0023] In certain aspects, the polyspecific polypeptide of the invention comprises a 4-1BB binding domain conjugated to a drug or toxin

[0024] The present invention also provides a polyspecific polypeptide comprising a 5T4 binding domain fused or conjugated to an immunoglobulin constant region. The immunoglobulin constant region may be a human immunoglobulin Fc domain. In some embodiments, the Fc domain of a human immunoglobulin comprises the sequence set forth in SEQ ID NO: 158 or SEQ ID NO: 160. In some embodiments, the present invention provides a multispecific polypeptide comprising, from the amino terminus to the carboxy terminus: (i) 5T4 -binding domain, (ii) hinge region, (iii) immunoglobulin constant region, (iv) linker binding domains, and (v) 4-1BB binding domain. In some embodiments, the linker of the binding domains comprises a Gly ₄ Ser sequence. In some examples, the linker of the binding domains contains a structure of the formula (Gly ₄ Ser) _n , where n = 1-5. In some aspects, the binding domain linker comprises an amino acid sequence selected from SEQ ID NO: 85-108. In certain embodiments, the binding domain linker comprises the amino acid sequence set forth in SEQ ID NO: 107 (SGGGGSGGGGSGGGGSPS).

[0025] In some aspects, the present invention also provides a polyspecific polypeptide comprising a first scFv domain and a second scFv domain, wherein said first and second scFv domains are linked to each other through a binding domain linker or a binding domain linker and an immunoglobulin Fc domain wherein said immunoglobulin Fc domain comprises a hinge region and an immunoglobulin constant region; and wherein said second scFv domain specifically binds to human 4-1BB and comprises: (i) an immunoglobulin heavy chain variable region comprising an HCDR1 amino acid sequence selected from the group consisting of SEQ ID NO: 2, 18, 24, an HCDR2 amino acid sequence , representing SEQ ID NO: 4, and the amino acid sequence of HCDR3, representing SEQ ID NO: 6; and (ii) an immunoglobulin light chain variable region comprising the amino acid sequence LCDR1 being SEQ ID NO: 8, the amino acid sequence LCDR2 being SEQ ID NO: 10, and the amino acid sequence LCDR3 being SEQ ID NO: 12, wherein the first and second scFv domains are linked to each other through a binding domain linker or a binding domain linker and an immunoglobulin Fc domain, wherein said immunoglobulin Fc domain comprises a hinge region and an immunoglobulin constant region. In certain embodiments, the polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity to a sequence selected from the group consisting of SEQ ID NO: 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, and 156. In some aspects, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, and 156. In some embodiments, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 136, 138, and 146.

[0026] In some embodiments, the polyspecific polypeptide comprises, in an amino-terminal to carboxy-terminal direction, (i) a first scFv domain, (ii) a linker binding domains, and (iii) a second binding domain. In other embodiments, the polyspecific polypeptide comprises, in an amino-terminal to carboxy-terminal direction, (i) a second scFv domain, (ii) a linker of binding domains, and (iii) a first scFv domain. In certain aspects, the polyspecific polypeptide comprises, from the amino terminus to the carboxy terminus: (i) a first scFv domain, (ii) a hinge region, (iii) an immunoglobulin constant region, (iv) a linker binding domain, and (v) a second scFv domain. In some embodiments, the linker of the binding domains comprises a Gly ₄ Ser sequence. In some embodiments, the linker of the binding domains comprises a structure of the formula (Gly ₄ Ser) _n , where n = 1-5. In certain aspects, the binding domain linker comprises an amino acid sequence selected from SEQ ID NO: 85-108. In certain embodiments, the binding domain linker comprises the amino acid sequence set forth in SEQ ID NO: 107 (SGGGGSGGGGSGGGGSPS). In some embodiments, the second scFv domain of the polyspecific polypeptide comprises: (a) the amino acid sequence of HCDR1 set forth in SEQ ID NO: 2; (b) the amino acid sequence of HCDR2 shown in SEQ ID NO: 4; (c) the amino acid sequence of HCDR3 shown in SEQ ID NO: 6; (d) the amino acid sequence of LCDR1 shown in SEQ ID NO: 8; (e) the amino acid sequence of LCDR2 shown in SEQ ID NO: 10; and (f) the amino acid sequence of LCDR3 set forth in SEQ ID NO: 12. In other embodiments, the second scFv domain of the polyspecific polypeptide comprises: (a) the amino acid sequence of HCDR1 set forth in SEQ ID NO: 24; (b) the amino acid sequence of HCDR2 shown in SEQ ID NO: 4; (c) the amino acid sequence of HCDR3 shown in SEQ ID NO: 6; (d) the amino acid sequence of LCDR1 shown in SEQ ID NO: 8; (e) the amino acid sequence of LCDR2 shown in SEQ ID NO: 10; and (f) the amino acid sequence of LCDR3 set forth in SEQ ID NO: 12. In some embodiments, the second scFv domain of the polyspecific polypeptide comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 14 and a light chain variable region comprising SEQ ID NO: 14 ID NO: 16. In other embodiments, the second scFv domain of the polyspecific polypeptide comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 20 and a light chain variable region comprising SEQ ID NO: 22. In some embodiments, the second scFv The -domain of the polyspecific polypeptide comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 26 and a light chain variable region of SEQ ID NO: 16. In some embodiments, the second scFv domain of the polyspecific polypeptide comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 28, and the light chain variable region of SEQ ID NO: 16.

[0027] The invention also relates to a polyspecific polypeptide comprising a first scFv domain and a second scFv domain, wherein said first and second scFv domains are linked to each other via a binding domain linker or a binding domain linker and an immunoglobulin Fc domain, wherein said Fc -immunoglobulin domain contains the hinge region and constant region of the immunoglobulin; wherein said second scFv domain specifically binds to human 4-1BB; and wherein said first scFv domain comprises (i) an immunoglobulin heavy chain variable region comprising an HCDR1 amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 52 and 60, an HCDR2 amino acid sequence selected from the group consisting of SEQ ID NO: 32 and 62, and the amino acid sequence of HCDR3, which is SEQ ID NO: 34; and (ii) an immunoglobulin light chain variable region comprising an amino acid sequence of LCDR1 selected from the group consisting of SEQ ID NOs: 8, 42 and 54, an amino acid sequence of LCDR2 of SEQ ID NO: 10, and an amino acid sequence of LCDR3 of SEQ ID NO: 36; and wherein said second scFv domain comprises (i) an immunoglobulin heavy chain variable region comprising an HCDR1 amino acid sequence selected from the group consisting of SEQ ID NO: 2, 18, 24, an HCDR2 amino acid sequence selected from SEQ ID NO: 4, and the amino acid sequence of HCDR3 represented by SEQ ID NO: 6; and (ii) an immunoglobulin light chain variable region comprising the amino acid sequence LCDR1 being SEQ ID NO: 8, the amino acid sequence LCDR2 being SEQ ID NO: 10, and the amino acid sequence LCDR3 being SEQ ID NO: 12, wherein the first and second scFv domains are linked together by a linker of binding domains or a linker of binding domains and an immunoglobulin Fc domain, wherein said immunoglobulin Fc domain comprises a hinge region and an immunoglobulin constant region. In some embodiments, the second scFv domain comprises (a) the amino acid sequence of HCDR1 set forth in SEQ ID NO: 2; (b) the amino acid sequence of HCDR2 shown in SEQ ID NO: 4; (c) the amino acid sequence of HCDR3 shown in SEQ ID NO: 6; (d) the amino acid sequence of LCDR1 shown in SEQ ID NO: 8; (e) the amino acid sequence of LCDR2 shown in SEQ ID NO: 10; and (f) the amino acid sequence of LCDR3 set forth in SEQ ID NO: 12. In certain embodiments, the first scFv domain comprises: (a) the amino acid sequence of HCDR1 set forth in SEQ ID NO: 30; (b) the amino acid sequence of HCDR2 shown in SEQ ID NO: 32; (c) the amino acid sequence of HCDR3 shown in SEQ ID NO: 34; (d) the amino acid sequence of LCDR1 shown in SEQ ID NO: 8; (e) the amino acid sequence of LCDR2 shown in SEQ ID NO: 10; and (f) the amino acid sequence of LCDR3 set forth in SEQ ID NO: 36. In some embodiments, the first scFv domain comprises: (a) the amino acid sequence of HCDR1 set forth in SEQ ID NO: 30; (b) the amino acid sequence of HCDR2 shown in SEQ ID NO: 32; (c) the amino acid sequence of HCDR3 shown in SEQ ID NO: 34; (d) the amino acid sequence of LCDR1 shown in SEQ ID NO: 42; (e) the amino acid sequence of LCDR2 shown in SEQ ID NO: 10; and (f) the amino acid sequence of LCDR3 set forth in SEQ ID NO: 36. In certain aspects, the first scFv domain comprises: (a) the amino acid sequence of HCDR1 set forth in SEQ ID NO: 30; (b) the amino acid sequence of HCDR2 shown in SEQ ID NO: 32; (c) the amino acid sequence of HCDR3 shown in SEQ ID NO: 34; (d) the amino acid sequence of LCDR1 shown in SEQ ID NO: 42; (e) the amino acid sequence of LCDR2 shown in SEQ ID NO: 10; and (f) the amino acid sequence of LCDR3 set forth in SEQ ID NO: 36. In some embodiments, the first scFv domain comprises: (a) the amino acid sequence of HCDR1 set forth in SEQ ID NO: 52; (b) the amino acid sequence of HCDR2 shown in SEQ ID NO: 32; (c) the amino acid sequence of HCDR3 shown in SEQ ID NO: 34; (d) the amino acid sequence of LCDR1 shown in SEQ ID NO: 54; (e) the amino acid sequence of LCDR2 shown in SEQ ID NO: 10; and (f) the amino acid sequence of LCDR3 set forth in SEQ ID NO: 36. In some embodiments, the first scFv domain comprises: (a) the amino acid sequence of HCDR1 set forth in SEQ ID NO: 60; (b) the amino acid sequence of HCDR2 shown in SEQ ID NO: 62; (c) the amino acid sequence of HCDR3 shown in SEQ ID NO: 34; (d) the amino acid sequence of LCDR1 shown in SEQ ID NO: 54; (e) the amino acid sequence of LCDR2 shown in SEQ ID NO: 10; and (f) the amino acid sequence of LCDR3 set forth in SEQ ID NO: 36. In certain embodiments, the second scFv domain comprises: (a) the amino acid sequence of HCDR1 set forth in SEQ ID NO: 18; (b) the amino acid sequence of HCDR2 shown in SEQ ID NO: 4; (c) the amino acid sequence of HCDR3 shown in SEQ ID NO: 6; (d) the amino acid sequence of LCDR1 shown in SEQ ID NO: 8; (e) the amino acid sequence of LCDR2 shown in SEQ ID NO: 10; and (f) the amino acid sequence of LCDR3 set forth in SEQ ID NO: 12. In some embodiments, the first scFv domain comprises: (a) the amino acid sequence of HCDR1 set forth in SEQ ID NO: 30; (b) the amino acid sequence of HCDR2 shown in SEQ ID NO: 32; (c) the amino acid sequence of HCDR3 shown in SEQ ID NO: 34; (d) the amino acid sequence of LCDR1 shown in SEQ ID NO: 42; (e) the amino acid sequence of LCDR2 shown in SEQ ID NO: 10; and (f) the amino acid sequence of LCDR3 set forth in SEQ ID NO: 36. In some embodiments, the second scFv domain comprises: (a) the amino acid sequence of HCDR1 set forth in SEQ ID NO: 24; (b) the amino acid sequence of HCDR2 shown in SEQ ID NO: 4; (c) the amino acid sequence of HCDR3 shown in SEQ ID NO: 6; (d) the amino acid sequence of LCDR1 shown in SEQ ID NO: 8; (e) the amino acid sequence of LCDR2 shown in SEQ ID NO: 10; and (f) the amino acid sequence of LCDR3 set forth in SEQ ID NO: 12. In some embodiments, the first scFv domain comprises: (a) the amino acid sequence of HCDR1 set forth in SEQ ID NO: 52; (b) the amino acid sequence of HCDR2 shown in SEQ ID NO: 32; (c) the amino acid sequence of HCDR3 shown in SEQ ID NO: 34; (d) the amino acid sequence of LCDR1 shown in SEQ ID NO: 54; (e) the amino acid sequence of LCDR2 shown in SEQ ID NO: 10; and (f) the amino acid sequence of LCDR3 set forth in SEQ ID NO: 36. In some embodiments, the second scFv domain comprises: (a) the amino acid sequence of HCDR1 set forth in SEQ ID NO: 24; (b) the amino acid sequence of HCDR2 shown in SEQ ID NO: 4; (c) the amino acid sequence of HCDR3 shown in SEQ ID NO: 6; (d) the amino acid sequence of LCDR1 shown in SEQ ID NO: 8; (e) the amino acid sequence of LCDR2 shown in SEQ ID NO: 10; and (f) the amino acid sequence of LCDR3 set forth in SEQ ID NO: 12, and optionally the first scFv domain comprises: (a) the amino acid sequence of HCDR1 set forth in SEQ ID NO: 30; (b) the amino acid sequence of HCDR2 shown in SEQ ID NO: 32; (c) the amino acid sequence of HCDR3 shown in SEQ ID NO: 34; (d) the amino acid sequence of LCDR1 shown in SEQ ID NO: 8; (e) the amino acid sequence of LCDR2 shown in SEQ ID NO: 10; and (f) the amino acid sequence of LCDR3 shown in SEQ ID NO: 36.

[0028] The present invention also provides a polyspecific polypeptide wherein the first scFv domain comprises i) a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 38 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 40, and wherein the second scFv domain comprises ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 14 and a light chain variable region comprising SEQ ID NO: 16. In some embodiments, the first scFv domain comprises i) a heavy chain variable region chain containing the amino acid sequence of SEQ ID NO: 38, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 44, and a second scFv domain comprising ii) a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 14, and a variable a light chain region comprising SEQ ID NO: 16. In certain embodiments, the first scFv domain comprises i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 46 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 46. 48, and the second scFv domain comprises ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 14, and a light chain variable region comprising SEQ ID NO: 16. In other embodiments, the first scFv domain comprises i) a variable a heavy chain region containing the amino acid sequence of SEQ ID NO: 56, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 58, and a second scFv domain comprising ii) a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 14, and a light chain variable region comprising SEQ ID NO: 16. In certain embodiments, the first scFv domain comprises i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 64 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 64. NO: 58, and the second scFv domain comprises ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 14 and a light chain variable region comprising SEQ ID NO: 16. In other embodiments, the first scFv domain comprises i ) a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 46, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 50, and a second scFv domain comprising ii) a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 50 20, and a light chain variable region comprising SEQ ID NO: 22. In other embodiments, the first scFv domain comprises i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 56, and a light chain variable region comprising the amino acid sequence SEQ ID NO: 58, and the second scFv domain comprises ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 26, and a light chain variable region comprising SEQ ID NO: 16. In some embodiments, the first scFv domain contains i) a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 38, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 68, and a second scFv domain contains ii) a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 28, and a light chain variable region comprising SEQ ID NO: 16. In some embodiments, the first scFv domain comprises i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 46, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 70, and the second scFv domain contains ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 28, and a light chain variable region comprising SEQ ID NO: 16.

[0029] In some embodiments, the present invention provides a polyspecific polypeptide comprising a first scFv domain and a second scFv domain, wherein said first and second scFv domains are linked to each other via a binding domain linker or a binding domain linker and an immunoglobulin Fc domain wherein said immunoglobulin Fc domain comprises a hinge region and an immunoglobulin constant region; and wherein said first scFv domain specifically binds to human 5T4 and comprises: (i) an immunoglobulin heavy chain variable region comprising the amino acid sequence HCDR1 being SEQ ID NO: 30, the amino acid sequence HCDR2 being SEQ ID NO: 32, and the amino acid sequence of HCDR3, which is SEQ ID NO: 34; and (ii) an immunoglobulin light chain variable region comprising the amino acid sequence LCDR1 of SEQ ID NO: 42, the amino acid sequence of LCDR2 of SEQ ID NO: 10, and the amino acid sequence of LCDR3 of SEQ ID NO: 36.

[0030] In some embodiments, the first scFv domain comprises: i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 38 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 44; or ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 46 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 48; or iii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 46 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 50.

[0031] In some embodiments, the present invention provides a polyspecific polypeptide comprising a first scFv domain and a second scFv domain, wherein said first and second scFv domains are linked to each other through a binding domain linker or a binding domain linker and an immunoglobulin Fc domain wherein said immunoglobulin Fc domain comprises a hinge region and an immunoglobulin constant region; and wherein said first scFv domain specifically binds to human 5T4, wherein the VH and/or VL region of said second scFv domain contains one or more mutations in the framework region; and wherein said first scFv domain comprises: (a) HCDR1 containing the amino acid sequence of SEQ ID NO: 30; (b) HCDR2 containing the amino acid sequence of SEQ ID NO: 32; (c) HCDR3 containing the amino acid sequence of SEQ ID NO: 34; (d) LCDR1 containing the amino acid sequence of SEQ ID NO: 42; (e) LCDR2 containing the amino acid sequence of SEQ ID NO: 10; and (f) LCDR3 comprising the amino acid sequence of SEQ ID NO: 36. In some embodiments, the first scFv domain comprises i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 38 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 44; or ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 46 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 48; or iii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 46 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 50.

[0032] In some embodiments, the polyspecific polypeptides described herein comprise, in an amino-terminal to carboxy-terminal direction, (i) a first scFv domain, (ii) a linker binding domains, and (iii) a second binding domain. In some embodiments, the polyspecific polypeptides comprise, in an amino-terminal to carboxy-terminal direction, (i) a second scFv domain, (ii) a linker of binding domains, and (iii) a first scFv domain. In some embodiments, the polyspecific polypeptides comprise, from the amino terminus to the carboxy terminus: (i) a first scFv domain, (ii) a hinge region, (iii) an immunoglobulin constant region, (iv) a linker binding domain, and (v) a second scFv domain . In some embodiments, the linker of the binding domains comprises a Gly ₄ Ser sequence. In some embodiments, the linker of the binding domains comprises a structure of the formula (Gly ₄ Ser) _n , where n = 1-5. In some embodiments, the binding domain linker comprises an amino acid sequence selected from SEQ ID NO: 85-108. In some embodiments, the binding domain linker comprises the amino acid sequence set forth in SEQ ID NO: 107 (SGGGGSGGGGSGGGGSPS).

[0033] In certain aspects, the first scFv domain of the polyspecific polypeptide of the invention specifically binds to human 5T4 and comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 118, 120, 122, 124, 126, 128, 130, 132, 134 and 170; and the second scFv domain specifically binds to human 4-1BB and comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 110, 112, 114, and 116. In some embodiments, the first scFv domain comprises an amino acid sequence set forth in SEQ ID NO: 118, and the second scFv domain contains the amino acid sequence set forth in SEQ ID NO: 110. In other embodiments, the first scFv domain contains the amino acid sequence set forth in SEQ ID NO: 120, and the second scFv domain contains the amino acid sequence set forth in SEQ ID NO: 110. In certain embodiments, the first scFv domain comprises the amino acid sequence set forth in SEQ ID NO: 122 and the second scFv domain contains the amino acid sequence set forth in SEQ ID NO: 110. In some embodiments the first scFv domain contains the amino acid sequence set forth in SEQ ID NO: 124, and the second scFv domain contains the amino acid sequence set forth in SEQ ID NO: 112. In some embodiments, the first scFv domain contains the amino acid sequence set forth in SEQ ID NO: : 126, and the second scFv domain contains the amino acid sequence set forth in SEQ ID NO: 114. In other embodiments, the first scFv domain contains the amino acid sequence set forth in SEQ ID NO: 126, and the second scFv domain contains the amino acid sequence set forth in in SEQ ID NO: 110. In certain embodiments, the first scFv domain contains the amino acid sequence set forth in SEQ ID NO: 128, and the second scFv domain contains the amino acid sequence set forth in SEQ ID NO: 110. In some embodiments, the first scFv -domain contains the amino acid sequence set forth in SEQ ID NO: 170, and the second scFv domain contains the amino acid sequence set forth in SEQ ID NO: 116. In some embodiments, the first scFv domain contains the amino acid sequence set forth in SEQ ID NO: 130 and the second scFv domain contains the amino acid sequence set forth in SEQ ID NO: 116. In some embodiments, the first scFv domain contains the amino acid sequence set forth in SEQ ID NO: 132, and the second scFv domain contains the amino acid sequence set forth in SEQ ID NO: 116. In other embodiments, the first scFv domain contains the amino acid sequence set forth in SEQ ID NO: 134, and the second scFv domain contains the amino acid sequence set forth in SEQ ID NO: 116. In some embodiments, the invention relates to a multispecific a polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity with a sequence selected from the group consisting of SEQ ID NO: 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 172, 174, and 176. In some embodiments, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 136, 138, 140, 142, 144 , 146, 148, 150, 152, 154, 156, 172, 174 and 176.

[0034] In some embodiments, the invention provides a polyspecific polypeptide that specifically binds 5T4 and 4-1BB, wherein the polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least at least 95% identity to a sequence selected from the group consisting of SEQ ID NOs: 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 172, 174, and 176. In certain embodiments, the polypeptide , specifically binding to 5T4 and 4-1BB, contains an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity to a sequence selected from the group consisting of SEQ ID NO: 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 172, 174 and 176, wherein said polypeptide contains the same CDR amino acid sequences as the corresponding SEQ ID NO:, or said the polypeptide contains CDR amino acid sequences that differ from the corresponding SEQ ID NO: by at most one amino acid. In some embodiments, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 172, 174, and 176.

[0035] The present invention also provides a multispecific polypeptide wherein the first scFv domain binds to the 5T4 extracellular domain, and where the second scFv domain binds to the 4-1BB extracellular domain. In some aspects, the polypeptide results in increased activation of effector cells. In some aspects, the polyspecific polypeptide of the invention increases effector cell activation and/or effector cell proliferation. In other aspects, the polyspecific polypeptide enhances effector cell-dependent lysis of 5T4-expressing cells. In some embodiments, the 5T4 binding domain of the invention is capable of binding 5T4 with a kD value of less than 50 nM. In some aspects, the light chain variable region and/or heavy chain variable region of the 5T4 binding domain and/or 4-1BB binding domain is humanized.

[0036] In some embodiments, the present invention provides a polyspecific polypeptide comprising a first single chain variable fragment (scFv) domain and a second scFv domain linked to each other by a linker of binding domains and an immunoglobulin Fc domain, wherein said Fc -immunoglobulin domain contains the hinge region and constant region of the immunoglobulin; wherein said polyspecific polypeptide is capable of forming a homodimer by association with a second identical polyspecific polypeptide; wherein said first scFv domain specifically binds to human 5T4 and comprises: (i) an immunoglobulin heavy chain variable region containing the amino acid sequence of the heavy chain complementarity determining region (HCDR)-1 representing SEQ ID NO: 30, the amino acid sequence of HCDR2 representing being SEQ ID NO: 32, and the amino acid sequence of HCDR3 being SEQ ID NO: 34; and (ii) an immunoglobulin light chain variable region comprising an amino acid sequence of a light chain complementarity determining region (LCDR)-1 being SEQ ID NO: 42, an amino acid sequence of LCDR2 being SEQ ID NO: 10, and an amino acid sequence of LCDR3 being is SEQ ID NO: 36; and wherein said second scFv domain specifically binds to human 4-1BB and comprises: (i) an immunoglobulin heavy chain variable region comprising the amino acid sequence of HCDR1 being SEQ ID NO: 2, the amino acid sequence of HCDR2 being SEQ ID NO: 4 , and the amino acid sequence of HCDR3, which is SEQ ID NO: 6; and (ii) an immunoglobulin light chain variable region comprising the amino acid sequence LCDR1 being SEQ ID NO: 8, the amino acid sequence LCDR2 being SEQ ID NO: 10, and the amino acid sequence LCDR3 being SEQ ID NO: 12. 2. The polyspecific polypeptide according to claim 1, containing, in the direction from the amino terminus to the carboxy terminus: (i) a first scFv domain, (ii) a hinge region, (iii) an immunoglobulin constant region, (iv) a linker binding domains and (v) a second scFv- domain. In some embodiments, the first scFv domain contains a mutation in the framework region relative to the framework region of SEQ ID NO: 130 or 170. In some embodiments, the mutation introduces a stabilizing disulfide bond.

[0037] In some embodiments, the first scFv domain of the polyspecific polypeptide comprises an immunoglobulin heavy chain variable region of SEQ ID NO: 38 and an immunoglobulin light chain variable region of SEQ ID NO: 44. In some embodiments, the first scFv- the domain of the polyspecific polypeptide comprises an immunoglobulin heavy chain variable region of SEQ ID NO: 46 and an immunoglobulin light chain variable region of SEQ ID NO: 48. In some embodiments, the second scFv domain of the polyspecific polypeptide comprises an immunoglobulin heavy chain variable region of being SEQ ID NO: 14, and an immunoglobulin light chain variable region being SEQ ID NO: 16. In some embodiments, the first scFv domain of the polyspecific polypeptide comprises an immunoglobulin heavy chain variable region being SEQ ID NO: 46, and a variable an immunoglobulin light chain variable region of SEQ ID NO: 48, and a second scFv domain comprising an immunoglobulin heavy chain variable region of SEQ ID NO: 14 and an immunoglobulin light chain variable region of SEQ ID NO: 16. B In some embodiments, the amino acid sequence of the first scFv domain is at least 97% identical to SEQ ID NO: 120, and the amino acid sequence of the second scFv domain is at least 97% identical to SEQ ID NO: 110. In some embodiments, the polypeptide comprises an amino acid sequence is at least 95% identical to the sequence of SEQ ID NO: 172, or contains an amino acid sequence identical to the sequence of SEQ ID NO: 172. In some embodiments, the amino acid sequence of the first scFv domain is at least 97% identical to SEQ ID NO: 122, and the amino acid sequence of the second scFv domain is at least 97% identical to SEQ ID NO: 110. In some embodiments, the polypeptide contains an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 174, or contains an amino acid sequence of SEQ ID NO: 174. 174.

[0038] In some embodiments, binding of the polyspecific polypeptide to an effector cell results in increased activation of the effector cell, increased proliferation of the effector cell, or binding of the polyspecific polypeptide to an effector cell and a 5T4-expressing cell results in enhanced effector cell-dependent lysis of said 5T4-expressing cell. .

[0039] The invention also includes a dimer comprising two identical polypeptides, wherein each of the two polypeptides is a polyspecific polypeptide as described herein.

[0040] In certain aspects, the disclosure of the invention relates to a pharmaceutical composition comprising a polypeptide or protein described herein and a pharmaceutically acceptable carrier, diluent or excipient. In some embodiments, the pharmaceutical composition may be provided in a dosage form selected from the group consisting of: a single dose oral form, a single dose intravenous form, a single dose intranasal form, a single dose suppository form, a single dose intradermal form, a single dose intramuscular form, single dose intraperitoneal form, single dose subcutaneous form, single dose epidural form, single dose sublingual form and single dose intracerebral form. Non-limiting examples of single dose oral form include tablets, pills, pellets, capsules, powders, lozenges, granules, solutions, suspensions, emulsions, syrups, elixirs, sustained release formulations, aerosols and sprays.

[0041] The invention also includes a method of enhancing effector cell activation directed against a 5T4-expressing cell, comprising: contacting said 5T4-expressing cell with a polypeptide or protein of the invention, wherein said contacting is carried out under conditions under which enhanced activation is induced effector cells directed against the specified 5T4-expressing cell. In some aspects, the invention provides a method of treating a disorder in a subject, wherein said disorder is characterized by 5T4 expression, comprising administering to said subject a therapeutically effective amount of a polypeptide or protein or pharmaceutical composition of the invention. The invention also includes the use of a polypeptide or protein of the invention for the manufacture of a medicament for treating a disorder in a subject wherein said disorder is characterized by 5T4 expression. In some embodiments, the invention provides a polypeptide or protein of the invention for use in treating a disorder in a subject wherein said disorder is characterized by 5T4 expression. In some embodiments, the disorder is cancer. In certain aspects, the cancer is breast cancer, pancreatic cancer, ovarian cancer, non-small cell lung cancer, mesothelioma, chronic lymphocytic leukemia (CLL), mantle cell leukemia (MCL), acute lymphoblastic leukemia (ALL), squamous cell carcinoma, melanoma, adrenal cancer , bladder cancer, cervical cancer, kidney cancer, stomach cancer, prostate cancer, thyroid cancer, liver cancer, uterine cancer, neurofibroma, sarcoma or head and neck cancer.

[0042] In some embodiments, a polyspecific polypeptide for use in such methods comprises, from the amino terminus to the carboxy terminus: (i) a first scFv domain, (ii) a hinge region, (iii) an immunoglobulin constant region, (iv) a linker binding domains and (v) a second scFv domain. In some embodiments, a polyspecific polypeptide for use in such methods comprises a second scFv domain comprising an immunoglobulin heavy chain variable region of SEQ ID NO: 14 and an immunoglobulin light chain variable region of SEQ ID NO: 16, and a first scFv -a domain comprising (i) an immunoglobulin heavy chain variable region of SEQ ID NO: 38 and an immunoglobulin light chain variable region of SEQ ID NO: 44; or (ii) an immunoglobulin heavy chain variable region of SEQ ID NO: 46 and an immunoglobulin light chain variable region of SEQ ID NO: 48. In some embodiments, the amino acid sequence of the second scFv domain of the polyspecific polypeptide is at least 97 % identical to SEQ ID NO: 110, and the amino acid sequence of the first scFv domain of the polyspecific polypeptide is at least 97% identical to SEQ ID NO: 120 (scFv to 5T4 for 209) or SEQ ID NO: 122. In some embodiments, the polypeptide exhibits statistically significantly enhanced effector cell activation compared to a second polyspecific polypeptide, wherein said second polyspecific polypeptide is an IgG-scFv structure comprising an anti-4-1BB antibody comprising a variable heavy chain comprising SEQ ID NO: 28 and a variable light chain containing SEQ ID NO: 16, and an anti-5T4 scFv comprising a variable heavy chain comprising SEQ ID NO: 46 and a variable light chain comprising SEQ ID NO: 66. In some embodiments, the polypeptide induces statistically significantly increased proliferation of effector cells compared with a second polyspecific polypeptide, wherein said second polyspecific polypeptide is an IgG-scFv structure comprising an anti-4-1BB antibody comprising a variable heavy chain comprising SEQ ID NO: 28 and a variable light chain comprising SEQ ID NO: 16, and a scFv against 5T4, containing a variable heavy chain containing SEQ ID NO: 46, and a variable light chain containing SEQ ID NO: 66.

[0043] Certain aspects of the invention include an isolated nucleic acid molecule encoding a polypeptide of the invention. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137 , 139, 141, 143, 145, 147, 149, 151, 153, 155, 171, 173 and 175. The invention includes an expression vector containing an isolated nucleic acid molecule described herein. In some embodiments, a nucleic acid molecule in an expression vector is operably linked to regulatory sequences suitable for expression of the nucleic acid segment in a host cell. The invention relates to a recombinant host cell containing an expression vector described herein. In some aspects, the invention provides a method for producing a polypeptide containing a 5T4 binding domain, comprising culturing a recombinant host cell containing an expression vector described herein under conditions under which the nucleic acid segment is expressed, thereby producing a polypeptide containing 5T4 -binding domain.

Brief description of graphic materials

[0044] In FIG. Figure 1 illustrates the agonistic function of seven constructs (ALG.APV-004, ALG.APV-006, ALG.APV-099, ALG.APV-127, ALG.APV-148 and ALG.APV-150) in the presence of 5T4(+) cells .

[0045] In FIG. Figure 2 illustrates the binding curves of seven scFv-Fc-scFv molecules (ALG.APV-006, ALG.APV-178, ALG.APV-179, ALG.APV-187, ALG.APV-191, ALG.APV-196 and ALG. APV-198) with the CHO-K1 cell line expressing human 4-1BB.

[0046] In FIG. Figure 3 illustrates the binding curves of seven scFv-Fc-scFv molecules (ALG.APV-006, ALG.APV-178, ALG.APV-179, ALG.APV-187, ALG.APV-191, ALG.APV-196 and ALG. APV-198) with the CHO-K1 cell line expressing cynomolgus 4-1BB.

[0047] In FIG. Figure 4 illustrates the binding curves of seven scFv-Fc-scFv molecules (ALG.APV-006, ALG.APV-178, ALG.APV-179, ALG.APV-187, ALG.APV-191, ALG.APV-196 and ALG. APV-198) with the CHO-K1 cell line expressing human 5T4.

[0048] In FIG. 5 illustrates the expression levels of human 5T4 in the CHO-K1 and SKOV-3 cell lines.

[0049] In FIG. Figure 6 illustrates the binding curves of constructs ALG.APV-006, ALG.APV-178, ALG.APV-179, ALG.APV-187, ALG.APV-196 and ALG.APV-198 on SKOV-3 cells.

[0050] In FIG. Figure 7 illustrates the binding curves of ALG.APV-006, ALG.APV-178, ALG.APV-179, ALG.APV-187, ALG.APV-191, ALG.APV-196 and ALG.APV-198 on CHO- line cells K1 expressing 5T4 cynomolgus monkeys.

[0051] In FIG. Figure 8 illustrates the activity of the constructs to 5T4 x 4-1BB in the 4-1BB reporter assay.

[0052] In FIG. Figure 9 illustrates the effect of the 5T4 x 4-1BB constructs on the release of IFN-γ from co-cultures of PBMC and CHO-K1.

[0053] In FIG. 10 illustrates an exemplary embodiment of a protein in the scFv-Fc-scFv format (ADAPTIR™ format).

[0054] In FIG. 11A - FIG. 11B shows the interferon-gamma (IFN-γ) response in human CD8+ T cells cultured with scFv-Fc-scFv bispecific constructs. In FIG. 11A shows T cell responses when cultured on 5T4-Fc antigen-coated plates. In FIG. 11B shows T cell responses when cultured without 5T4-Fc antigen. The figure shows the average values for two donors.

[0055] In FIG. 12 shows the binding curves of ALG.APV-004, ALG.APV-178, ALG.APV-179, ALG.APV-187, ALG.APV-208, ALG.APV-209 and ALG.APV-210 with the CHO- cell line K1/ CD137 human.

[0056] In FIG. Figure 13 shows the binding curves of ALG.APV-004, ALG.APV-178, ALG.APV-179, ALG.APV-187, ALG.APV-208, ALG.APV-209 and ALG.APV-210 to mouse CT26 cells ( ATCC) expressing human 5T4.

[0057] In FIG. 14 shows the binding curves of ALG.APV-004, ALG.APV-178, ALG.APV-179, ALG.APV-187, ALG.APV-208, ALG.APV-209 and ALG.APV-210 on CHO-K1 transfectants /5T4 cynomolgus macaque.

[0058] In FIG. 15 shows the binding curves of ALG.APV-004, ALG.APV-178, ALG.APV-179, ALG.APV-187, ALG.APV-208, ALG.APV-209 and ALG.APV-210 in a human cell line, expressing neither CD137 nor 5T4 (MOLM13, ATCC cells).

[0059] In FIG. 16 shows the activity of bispecific constructs in Jurkat/NF-κB reporter cells after 5 hours of incubation in the presence of 5T4(+) cells (HCC1143) or 5T4(-) cells (MOLM13). Each point on the curve represents the average of parallel wells. The y-axis shows values in relative fluorescence units (RLU).

[0060] In FIG. 17 shows the levels of IFN-γ induced in primary PBMC cultures after 72 hours by ALG.APV-178, ALG.APV-179, ALG.APV-187, ALG.APV-208, ALG.APV-209, ALG.APV- 210, and a control design in the Morrison format, ALG.APV-004. Each point on the curve represents the average of parallel wells.

[0061] In FIG. 18A - FIG. 18B illustrates FACS analysis of 5T4-dependent localization of the bispecific constructs ALG.APV-209, ALG.APV-210 and ALG.APV-004 to antigen-presenting tumors. Localization was determined by staining with anti-human Fc (FIG. 18A) or biotinylated 4-1BB (FIG. 18B).

[0062] In FIG. 19A - FIG. 19B illustrates immunohistochemical analysis of 5T4-dependent localization of bispecific constructs ALG.APV-209, ALG.APV-210 and ALG.APV-004 in 5T4+ (FIG. 19A) and 5T4- (FIG. 19B) tumors.

[0063] In FIG. 20 shows the IFN-γ response of human CD8 T cells cultured with the bispecific antibody ALG.APV-210 in the presence of CT26 cells expressing varying levels of 5T4.

[0064] In FIG. 21A - FIG. 21B shows the maximal release of IFN-γ by human CD8 T cells cultured with the bispecific antibody ALG.APV-210 in the presence of CT26 cells expressing varying levels of 5T4.

[0065] In FIG. 22A - FIG. 22C shows the binding of ALG.APV-210 or ANC107 (isotype control) to primary CD8 T cells selected from CD3 stimulated or unstimulated human and cynomolgus PBMCs. MFI (and SEM) from 2 independent experiments are shown in FIG. 22A and FIG. 22B. In FIG. 22C shows normalized pooled data for ALG.APV-210 from 2 independent experiments. n = 3 donors/group/experiment.

[0066] In FIG. 23A - FIG. 23C shows the binding of ALG.APV-210 or ANC107 (isotype control) to primary CD8 T cells gated from CD3-stimulated or unstimulated human and cynomolgus PBMCs. The percentage and SEM of ALG.APV-210 binding to CD8 T cells from 2 independent experiments are shown in FIG. 23A and 23B. In FIG. Figure 23C shows normalized pooled data from Experiments 1 and 2. n = 3 donors/group/experiment.

[0067] In FIG. 24 shows the agonist function of ALG.APV-210 on human CD8 T cells.

[0068] In FIG. 25A - FIG. 25B shows the agonist function of ALG.APV-210 on human CD8 T cells from selected representative donors.

[0069] In FIG. 26 shows the agonist function of ALG.APV-210 on cynomolgus CD8 T cells.

[0070] In FIG. 27A - FIG. 27C shows the agonist function of ALG.APV-210 on cynomolgus CD8 T cells from selected representative donors. The figures show the dose-dependent production of IFN-γ by cynomolgus CD8 T cells activated with ALG.APV-210 in the presence of 5T4-Fc.

[0071] In FIG. 28 shows the antitumor efficacy of the bispecific antibody ALG.APV-210 in an HCT-116 - 5T4-positive xenograft model of human colon carcinoma in SCID beige mice.

[0072] In FIG. Figure 29 shows an analysis of the PK properties of ALG.APV-210 or ALG.APV-209.

[0073] In FIG. 30A - FIG. 30B shows the binding of the Fc portion of ALG.APV-210 to FcγR. Titred ALG.APV-210 was incubated with FcγR-expressing cells and probed with a fluorescently labeled anti-human IgG secondary antibody (FIG. 30A). A human IgG1 molecule was used as a positive control (FIG. 30B).

[0074] In FIG. 31A - FIG. 31B shows the proliferation of CD8+ and CD4+ T cells induced by the bispecific molecules ALG.APV-209 and ALG.APV-210 in the presence of CHO-K1 cells expressing human 5T4 and anti-CD3 antibodies.

[0075] In FIG. 32A - FIG. 32B shows the induction of IFN-γ secretion by the bispecific molecule ALG.APV-210 in whole blood PBMC cultures from two different human PBMC donors (A and B).

[0076] In FIG. 33A - FIG. 33B shows CD8+ T cell proliferation induced by the bispecific molecule ALG.APV-210 in whole blood PBMC cultures from two different human PBMC donors.

[0077] In FIG. 34 shows the binding of ALG.APV-210 to 5T4-expressing cell lines.

[0078] In FIG. 35 shows the agonist function of the ALG.APV-210 bispecific construct in Jurkat/NF-κB reporter cells after incubation in the presence of cells expressing a range of densities of 5T4 protein on their surface.

[0079] In FIG. 36 shows the IFN-γ response of human CD8+ T cells cultured with the bispecific antibody ALG.APV-210 in the presence of human HCT116 cells expressing 5T4.

[0080] In FIG. 37 shows the IFN-γ response of human CD8+ T cells cultured with the bispecific antibody ALG.APV-210 in the presence of human HCT116 cells expressing 5T4.

[0081] In FIG. 38 shows the maximal release of IFN-γ by human CD8+ T cells cultured with the bispecific antibody ALG.APV-210 in the presence of HCT116 cells expressing human 5T4.

[0082] In FIG. 39A - FIG. 39D shows the binding of ALG.APV-210 to human tumor cells expressing 5T4 or transfected cell lines.

[0083] In FIG. 40A - FIG. 40D shows normalized MFI for experiments demonstrating binding of ALG.APV-210 to 5T4-expressing human tumor cells or transfected cell lines.

Detailed Description of the Invention

[0084] The present invention relates to polypeptides containing binding domains that specifically bind to trophoblastic glycoprotein (5T4) and polypeptides that specifically bind to 5T4. In some embodiments, the polypeptides are polyspecific polypeptides capable of specifically binding to 5T4 and another target. In some embodiments, the polypeptides described herein are polyspecific polypeptides that specifically bind to 5T4 and also specifically bind to a target on an effector cell. The invention also relates to polypeptides containing binding domains that specifically bind to member 9 of the tumor necrosis factor receptor superfamily (41BB or CD137), and polypeptides that specifically bind to 4-1BB. In some embodiments, the polypeptides are polyspecific polypeptides capable of specifically binding to 4-1BB and another target (eg, a tumor-associated antigen). In some embodiments, the polypeptides described herein are polyspecific polypeptides that specifically bind to 4-1BB and also specifically bind to a target on a target cell. In some embodiments, the polyspecific polypeptides are bispecific polypeptides that specifically bind to 5T4 and specifically bind to 4-1BB. In some embodiments, the bispecific polypeptides bind to 5T4 expressed on a target cell and 4-1BB expressed on an effector cell, thereby resulting in increased activation of the effector cells and enhancing effector cell-mediated cytotoxicity to the target cell.

[0085] In some embodiments, the present invention provides 5T4 binding domains and/or 4-1BB binding domains (and polypeptides or proteins containing such binding domains) exhibiting less off-target binding compared to previously known 5T4 binding domains and /or 4-1BB binding domains. In certain aspects, the binding domains and/or binding domain-containing polypeptides described herein bind 5T4 and/or 4-1BB more efficiently in certain formats and/or certain orientations (e.g., V _H -V _L versus V _L -V _H ), resulting in higher potency and/or improved efficacy in the treatment of disorders associated with 5T4 expression.

[0086] In some embodiments, administration of a therapeutically effective amount of a polypeptide or protein described herein to a patient in need thereof is effective for treating certain disorders associated with 5T4 expression, including certain types of cancer. In one embodiment, the polypeptide or protein binds both a 5T4-expressing target cell and an effector cell, thereby linking the 5T4-expressing target cell and the effector cell. Binding of both domains to their targets enhances effector cell activation, leading to a longer-lasting and/or more sustained effector cell response (eg, effector cell-mediated cytotoxicity). The polypeptides and proteins of the present invention provide various benefits in the treatment of patients, for example, effective binding to 5T4, effective enhancement of effector cell activity, reduced levels of cytokine release and/or lower risk of adverse events (eg, toxicity). In some embodiments, the target cell expresses 5T4 at a higher level than the non-target cell (eg, a normal cell or a non-cancerous cell from the same subject, organ, or tissue). In other embodiments, the target cell expresses 5T4 while the non-target cell (eg, a normal cell or a non-cancerous cell from the same subject, organ, or tissue) does not express 5T4.

[0087] The section headings used herein are for organization purposes only and should not be construed as limiting the invention described. All documents or portions of documents cited herein, including, but not limited to, patents, patent applications, articles, books and treatises, are hereby expressly incorporated by reference in their entirety for all purposes. In the event that one or more of the included documents or portions of documents defines a term that is inconsistent with the definition of that term in the application, the definition given in this application shall control. However, reference to any source, article, publication, patent, patent publication and patent application cited herein is not and should not be construed as an admission or any form of suggestion that it constitutes relevant prior art or constitutes part of the public knowledge in any country in the world.

[0088] As used herein, any concentration range, percentage range, ratio range, or integer range should be understood to include the value of any integer within the specified range and, when applicable, fractions thereof (such as one-tenth and one-hundredth of an integer) if not otherwise stated. It should be understood that the singular terms used herein refer to “one or more” of the listed components unless otherwise indicated. The application of an alternative (such as “or”) should be understood to mean one, both, or any combination of these alternatives. As used herein, the terms “include” and “comprise” are synonymous. In addition, it should be understood that polypeptides containing various combinations of components (eg, domains or regions) and substituents described herein are disclosed herein to the same extent as if each polypeptide were described individually. Thus, the selection of specific components of individual polypeptides is within the scope of the present invention.

Definitions

[0089] As used herein, the term “binding domain” or “binding region” refers to a domain, region, portion, or site of a protein, polypeptide, oligopeptide, peptide, antibody, or antibody-derived binding domain having the ability to specifically recognize and bind to a target molecule such as an antigen, ligand, receptor, substrate or inhibitor (eg, 5T4 or 4-1BB). Exemplary binding domains include antibodies and antibody-like proteins or domains, antibody heavy and light chain variable regions, and single chain antibody variable regions (eg, domain antibodies, sFv, scFv, scFab). In certain embodiments, the binding domain comprises or consists of an antigen binding site (e.g., comprising a heavy chain variable sequence and a light chain variable sequence, or three light chain complementarity determining regions (CDRs) and three heavy chain CDRs of an antibody placed on alternative framework regions (FRs) ) (eg, human FRs, optionally containing one or more amino acid substitutions)). Various assays are known to identify binding domains of the present invention that specifically bind a particular target, including Western blotting, ELISA, phage display library screening, and BIACORE® interaction assay. In some embodiments, the polypeptides of the present invention comprise a binding domain that specifically binds to a target antigen expressed by the target cell (eg, a tumor-associated antigen such as 5T4). In some embodiments, the polypeptides of the present invention comprise a binding domain that specifically binds to a target antigen expressed by an effector cell (eg, 4-1BB). In some embodiments, the polypeptides of the present invention are polyspecific polypeptides and contain two or more binding domains.

[0090] A binding domain or protein containing a binding domain “specifically binds” a target if it binds said target with an affinity or K _a (that is, the equilibrium association constant of a particular binding interaction expressed in 1/M) equal to or greater than 10 ⁵ M ^-1 , and at the same time to a small extent binds other components present in the test sample. Binding domains can be classified as "high affinity" and "low affinity" binding domains. “High affinity” binding domains refer to binding domains whose K _a is at least 10 ⁷ M ^-1 , at least 10 ⁸ M ^-1 , at least 10 ⁹ M ^-1 , at least 10 ¹⁰ M ^-1 , at least 10 ¹¹ M ^-1 , at least 10 ¹² M ^-1 or at least 10 ¹³ M ^-1 . “Low affinity” binding domains refer to binding domains whose K _a is up to 10 ⁷ M ^-1 , up to 10 ⁶ M ^-1 , up to 10 ⁵ M ^-1 . Alternatively, affinity can be defined as the equilibrium dissociation constant (K _d ) of a particular binding interaction, expressed in M (e.g., 10 ^-5 M to 10 ^-13 , or about 500 nM, about 300 nM, about 250 nM, about 200 nM, about 150 nM, about 100 nM, about 50 nM, about 25 nM, about 10 nM, or about 5 nM). The binding domain affinities of the polypeptides and single chain polypeptides of the present invention can be readily determined using conventional techniques (see, for example, Scatchard et al. (1949) Ann. NY Acad. Sci. 51:660; and US Pat. Nos. 5,283,173, 5,468,614 , or their equivalent).

[0091] As used herein, the term “conservative substitution” is known in the art as the replacement of one amino acid with another amino acid having similar properties. Typical conservative substitutions are well known in the art (see, for example, PCT Application Publication No. WO 97/09433, page 10, published March 13, 1997; Lehninger, Biochemistry, Second Edition; Worth Publishers, Inc. NY:NY (1975), pp. 71-77; Lewin, Genes IV, Oxford University Press, NY and Cell Press, Cambridge, MA (1990), p. 8).

[0092] As used herein, the term “derivative” refers to modification of one or more amino acid residues of a peptide by chemical or biological means, with or without the use of an enzyme, such as glycosylation, alkylation, acylation, ester formation, or amide formation.

[0093] As used herein, a polypeptide or amino acid sequence “derived from” the specified polypeptide or protein refers to the origin of the polypeptide. In certain embodiments, a polypeptide or amino acid sequence derived from a specific sequence (sometimes referred to as the "original" or "parent" sequence) and having an amino acid sequence substantially identical to the original sequence or a portion thereof, where the portion consists of at least 10-20 amino acids , at least 20-30 amino acids, or at least 30-50 amino acids, or at least 50-150 amino acids, or otherwise can be identified by one skilled in the art as being derived from the original sequence. For example, a binding domain (eg, Fab, F(ab')2, Fab', scFv, single domain antibody (sdAb), etc.) can be derived from an antibody. In some embodiments, the binding domain sequence (eg, 5T4 or 4-1BB binding domain) is derived from the antibody or protein by a computer algorithm or in silico .

[0094] Polypeptides derived from another polypeptide may have one or more mutations or changes relative to the parent polypeptide, for example, one or more amino acid residues that have been replaced by another amino acid residue, or having one or more amino acid insertions or deletions. In such embodiments, polypeptides derived from a parent polypeptide and containing one or more mutations or changes are referred to as “variants.” As used herein, the term “variant” or “variants” refers to a polynucleotide or polypeptide having a sequence that differs from the sequence of the reference polynucleotide or polypeptide but retains its essential properties. In general, variant polynucleotide or polypeptide sequences are generally very similar and in many areas identical to the reference polynucleotide or polypeptide. For example, a variant polynucleotide or polypeptide may have at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the active moiety or full-length reference polynucleotide or polypeptide. The polypeptide may contain an amino acid sequence that does not occur in nature. Such variations necessarily have less than 100% sequence identity or similarity to the original polypeptide. In one embodiment, the variant will have an amino acid sequence having from about 60% to less than 100% identity or similarity to the amino acid sequence of the parent polypeptide. In yet another embodiment, the variant will have an amino acid sequence having from about 75% to less than 100%, from about 80% to less than 100%, from about 85% to less than 100%, from about 90% to less than 100 %, from about 95% to less than 100% identity or similarity to the amino acid sequence of the parent polypeptide.

[0095] As used herein, the term “sequence identity” refers to the relationship between two or more polynucleotide sequences or between two or more polypeptide sequences. When a position in one sequence is occupied by the same nucleic acid base or amino acid residue as the corresponding sequence position in the comparative sequence, the sequences are said to be “identical” at that position. Percent sequence identity is calculated by determining the number of positions at which an identical nucleic acid base or amino acid residue occurs in both sequences, yielding the number of identical positions. The number of identical positions is then divided by the total number of positions in the comparison window and multiplied by 100 to obtain the percent sequence identity. The percentage of sequence identity is determined by comparing two optimally aligned sequences in a comparison window. The comparison window for polynucleotide sequences may have a length of, for example, at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 or more nucleic acids. The comparison window for polypeptide sequences may have a length of, for example, at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300 or more amino acids. To optimally align sequences for comparison, a portion of the polynucleotide or polypeptide sequence in the comparison window may contain additions or deletions, called gaps, while the reference sequence is unchanged. An optimal alignment is one that, even with gaps, provides the maximum possible number of “identical” positions between the reference and reference sequences. The percentage of "sequence identity" between two sequences can be determined using the version of the "BLAST 2 Sequences" program available from the National Center for Biotechnology Information as of September 1, 2004, which includes the programs BLASTN (for comparing nucleotide sequences) and BLASTP (for comparing polypeptide sequences) based on the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90(12):5873-5877, 1993). When using "BLAST 2 Sequences", the parameters that were the default parameters on September 1, 2004 can be used for segment length (3), gap penalty (11), gap extension penalty (1), gap blowout ( 50), expected value (10), and any other required parameter, including but not limited to the matrix option. Two nucleotide or amino acid sequences are considered to have "substantially similar sequence identity" or are "substantially identical" if the two sequences share at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to each other.

[0096] As used herein, unless otherwise indicated, the position of an amino acid residue in the variable region of an immunoglobulin molecule is numbered according to IMGT criteria (Brochet et al. , Nucl. Acids Res. (2008) 36, W503-508), or in in accordance with EU nomenclature (Ward et al. , 1995 Therap. Immunol. 2:77-94), and the position of the amino acid residue in the constant region of the immunoglobulin molecule is numbered in accordance with EU nomenclature (Ward et al. , 1995 Therap. Immunol. 2: 77-94). The Kabat numbering system (Kabat, Sequences of Proteins of Immunological Interest, ^5th ed. Bethesda, MD: Public Health Service, National Institutes of Health (1991)) is an alternative system used to designate the position of an amino acid residue in the variable region of an immunoglobulin molecule, and in some cases it is used to indicate the position of an amino acid residue in the variable region of an immunoglobulin molecule herein.

[0097] As used herein, the term "dimer" refers to a biological entity that consists of two subunits linked to each other through one or more types of intramolecular forces, including covalent bonds (eg, disulfide bonds) and other interactions (eg, electrostatic interactions, salt bridges, hydrogen bonds and hydrophobic interactions), and is stable under appropriate conditions (e.g., under physiological conditions, in an aqueous solution suitable for the expression, purification and/or storage of recombinant proteins, or under conditions for non-denaturing and/or non-reducing electrophoresis). As used herein, the terms “heterodimer” or “heterodimeric protein” refer to a dimer formed from two different polypeptides. The heterodimer may contain a 5T4 x 4-1BB molecule, as described herein. The heterodimer does not include an antibody formed from four polypeptides (ie, two light chains and two heavy chains). The terms "homodimer" or "homodimer protein" as used herein refer to a dimer formed from two identical polypeptides.

[0098] "Fc region" or "Fc domain" refers to a polypeptide sequence corresponding to or derived from a portion of the parent antibody capable of binding to Fc receptors on cells and/or complement component C1q, thereby mediating the effector function of the antibody. Fc stands for "crystallizable fragment" - a fragment of an antibody that readily forms a protein crystal. Individual protein fragments, which were originally described by proteolytic cleavage, may determine the overall structure of the immunoglobulin protein. As originally defined in the literature, the Fc region is a homodimeric protein containing two polypeptides linked by disulfide bonds, each containing a hinge region, a CH2 domain and a CH3 domain. More recently, however, the term has been applied to a single-chain monomer component consisting of CH3, CH2 and at least a portion of a hinge sufficient to form a disulfide-linked dimer with a second such chain. For this reason, depending on the context, the use of the terms “Fc region” or “Fc domain” herein refers to either the dimeric form or the individual monomers associated to form a dimeric protein. For a review of the structure and function of immunoglobulins, see Putnam, The Plasma Proteins , Vol. V (Academic Press, Inc., 1987), pp. 49-140; and Padlan, Mol. Immunol . 31:169-217, 1994. As used herein, the term Fc includes variants of naturally occurring sequences.

[0099] “Immunoglobulin constant region” or “constant region” is a term defined herein to mean a peptide or polypeptide sequence derived from, or corresponding to, part or all of the sequence of one or more immunoglobulin constant domains. In certain embodiments, the constant region comprises the CH2 and CH3 domains of an IgG, for example, the CH2 and CH3 domains of an IgG1. In certain embodiments, the constant region does not contain a CH1 domain. In certain embodiments, the constant domains comprising the constant region are a human immunoglobulin region. In some embodiments (e.g., certain variations of a 41BB binding polypeptide, a 5T4 binding polypeptide, or polyspecific polypeptides containing them), the constant region of the fusion protein of the present invention lacks or has minimal effector functions while retaining the ability to bind certain Fc receptors , such as the neonatal Fc receptor (FcRn), and maintaining a relatively long half-life in vivo . For example, the constant region of the fusion protein of the present invention does not result in or significantly reduce the induction of antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), complement activation, and/or complement-dependent cytotoxicity (CDC). In other variations, the fusion protein of the present invention contains constant domains that retain one or more effector functions, for example, one or both of ADCC and CDC. In certain embodiments, the binding domain of the present invention is fused to a human IgG1 constant region, wherein the IgG1 constant region has one or more mutations of the following amino acids: leucine at position 234 (L234), leucine at position 235 (L235), glycine at position 237 ( G237), glutamate at position 318 (E318), lysine at position 320 (K320), lysine at position 322 (K322), or any combination thereof (EU numbering). For example, any one or more of these amino acids may be replaced by alanine. In a further embodiment, in the Fc domain of IgG1, L234, L235, G237, E318, K320, and K322 (as defined by EU numbering) are each mutated to alanine (i.e., L234A, L235A, G237A, E318A, K320A, and K322A, respectively) and, optionally, it also contains the N297A mutation (i.e., essentially eliminating glycosylation of the CH2 domain).

[00100] The terms "light chain variable region" (also referred to as "light chain variable domain" or " _VL ") and "heavy chain variable region" (also referred to as "heavy chain variable domain" or " _VH ") refer to variable binding region from the light and heavy chain of the antibody, respectively. Variable binding regions are composed of discrete, well-defined subregions known as “complementarity determining regions” (CDRs) and “framework regions” (FRs). In one embodiment, the FRs are humanized. The term "CL" refers to an "immunoglobulin light chain constant region" or a "light chain constant region", that is, a constant region from the light chain of an antibody. The term "CH" refers to the "immunoglobulin heavy chain constant region" or "heavy chain constant region", which is further subdivided, depending on the isotype of the antibody, into CH1, CH2 and CH3 domains (IgA, IgD, IgG), or CH1, CH2 , CH3 and CH4 (IgE, IgM). A "Fab" (antigen-binding fragment) is the antigen-binding portion of an antibody and includes a variable region and a heavy chain CH1 domain linked to a light chain via an interchain disulfide bond.

[00101] As used herein, the term “linker” generally refers to a short polypeptide sequence connecting two subdomains of a polypeptide. Non-limiting examples of linkers include flexible linkers containing glycine-serine repeats and linkers derived from (a) the interdomain region of a transmembrane protein (eg, type I transmembrane protein); (b) the stem region of a group II C-type lectin; or (c) an immunoglobulin hinge. In some embodiments, the linker provides a spacer function compatible with the interaction of two subbinding domains such that the resulting polypeptide retains specific binding affinity for the same target molecule as the antibody, which contains the same light and heavy chain variable regions. In certain embodiments, the linker contains from five to about 35 amino acids, such as from about 15 to about 25 amino acids. As used herein, the term “linker between CH3 and CH1 or CL” refers to one or more amino acid residues (e.g., about 2-12, about 2-10, about 4-10, about 5-10, about 6-10, about 7-10, about 8-10, about 9-10, about 8-12, about 9-12, or about 10-12) between the C-terminus of the CH3 domain (e.g., wild-type or mutated CH3) and the N-terminus of the domain CH1 or CL domain (eg Cκ).

[00102] In some embodiments, depending on the context, the linker may refer to (1) the polypeptide region between the V _H and V _L regions in a single chain Fv (scFv) or (2) the polypeptide region between the first binding domain and the second binding domain in a polyspecific polypeptide containing two binding domains. In the latter example, when a linker connects two or more binding domains, such a linker is referred to herein as a “binding domain linker.” In some embodiments, a binding domain linker may directly link or connect two or more binding domains, resulting in a construct comprising the following structure: binding domain - binding domain linker - binding domain. In some embodiments, the polyspecific polypeptides described herein comprise, in order from amino terminus to carboxy terminus, (i) a first binding domain, (ii) a linker binding domains, and (iii) a second binding domain. In some embodiments, the polyspecific polypeptide comprises, in order from amino terminus to carboxy terminus, (i) a second binding domain, (ii) a linker of binding domains, and (iii) a first binding domain. In some embodiments, a binding domain linker can bind or connect two or more binding domains by linking at least one binding domain to a non-binding domain polypeptide, such as an immunoglobulin Fc domain (i.e., a polypeptide comprising the structure: Ig hinge - Ig constant region ). In such embodiments, the resulting constructs may comprise the following structure: binding domain - Fc domain - linker binding domains - binding domain. In some embodiments, the polyspecific polypeptides described herein comprise, in order from amino terminus to carboxy terminus: (i) a first binding domain, (ii) a hinge region, (iii) an immunoglobulin constant region, (iv) a linker binding domains, and (v ) second binding domain. In some embodiments, the polyspecific polypeptide comprises, in order from amino terminus to carboxy terminus, (i) a second binding domain, (ii) a linker of binding domains, (iii) an immunoglobulin constant region, (iv) a hinge region, and (v) a first binding domain. The polypeptide region between the immunoglobulin constant region and the second binding domain in a polyspecific polypeptide containing two binding domains (e.g., a linker binding domains) may also be referred to as a “carboxy-terminal linker” or “amino-terminal linker,” depending on the orientation of the domains in the polyspecific polypeptide molecule. Non-limiting examples of linkers are shown in Table 1.

[00103] In some embodiments, “hinge” or “hinge region” refers to a polypeptide derived from the hinge region of an immunoglobulin and located in a polypeptide as described herein between a binding domain (e.g., a 5T4 binding domain or a 4-1BB binding domain) and immunoglobulin constant region. "Wild-type immunoglobulin hinge region" refers to the naturally occurring amino acid sequences of the upper and middle hinges located between and connecting the CH1 and CH2 domains (for IgG, IgA and IgD), or located between the CH1 and CH3 domains (for IgE and IgM) and connecting them, located in the heavy chain of the antibody. In certain embodiments, the wild-type immunoglobulin hinge sequence is human and may comprise a human IgG hinge region (eg, an IgG1, IgG2, IgG3, or IgG4 hinge region).

[00104] “Altered immunoglobulin hinge region” or “variant immunoglobulin hinge region” refers to a hinge region polypeptide with one or more mutations, substitutions, insertions or deletions compared to the corresponding original wild-type immunoglobulin hinge region. In certain embodiments, the altered immunoglobulin hinge region is at least 70% homologous to the wild-type immunoglobulin hinge region (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98 homologous) % or 99%). In certain embodiments, the altered immunoglobulin hinge region is a fragment of a wild-type immunoglobulin hinge region having a length of about 5 amino acids (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, 20 or more amino acids) to about 120 amino acids (e.g., having a length of from about 10 to about 40 amino acids, or from about 15 to about 30 amino acids, or from about 15 to about 20 amino acids, or from about 20 to approximately 25 amino acids). Typically, an altered immunoglobulin hinge region that is a fragment of a wild-type immunoglobulin hinge region comprises an IgG core hinge region (e.g., a polypeptide containing the sequence C-X-X-C, where X is any amino acid), as disclosed in US Patent Application Publication Nos. 2013/ 0129723 and 2013/0095097. Non-limiting examples of hinges are shown in Table 1.

[00105] As used herein, the term “humanized” refers to the process of rendering antibodies or immunoglobulin-binding proteins and polypeptides derived from a non-human species (e.g., mouse or rat) less immunogenic to humans while retaining the antigen-binding properties of the original antibodies using genetic engineering methods. In some embodiments, the binding domain(s) of the antibody or immunoglobulin binding proteins and polypeptides (eg, light and heavy chain variable regions, Fab, scFv) are humanized. Non-human binding domains can be humanized using methods known as CDR grafting (Joneset al., Nature 321:522 (1986)), and their variants, including "reconstruction" (Verhoeyen,et al., 1988 Science 239:1534-1536; Riechmann,et al., 1988 Nature 332:323-337; Tempest,et al., Bio/Technol 1991 9:266-271), “hyperchimerization” (Queen,et al., 1989 Proc Natl Acad Sci USA 86:10029-10033; Co,et al., 1991 Proc Natl Acad Sci USA 88:2869-2873; Co,et al., 1992 J Immunol 148:1149-1154) and “veining” (Mark,et al., “Derivation of therapeutically active humanized and veneered anti-CD18 antibodies.” B: Metcalf BW, Dalton BJ, eds. Cellular adhesion: molecular definition to therapeutic potential (New York: Plenum Press, 1994: 291-312).Other regions of antibodies or immunoglobulin-binding proteins and polypeptides, such as the hinge region and constant region domains, if derived from a non-human source, they may also be humanized.The knowledge of humanized antibodies in the art applies to the polypeptides of the invention even if the polypeptides are not antibodies.

[00106] As used herein, the term “patient in need” refers to a patient at risk of developing a disease, disorder, or condition that is amenable to treatment or amelioration with a 5T4 binding protein or polyspecific polypeptide, or a composition containing the same, as provided herein , or suffering from such a disease, disorder or condition.

[00107] As used herein, the term “pharmaceutically acceptable” refers to chemical compounds and compositions that do not typically cause allergic or other serious adverse reactions when administered by methods well known in the art. Chemical compounds and compositions that are approved by a regulatory agency of the Federal or State government, or listed in the United States Pharmacopoeia or other generally accepted pharmacopoeia for use in animals and, more specifically, in humans, are considered “pharmaceutically acceptable.”

[00108] As used herein, the term “promoter” refers to a region of DNA involved in binding RNA polymerase to initiate transcription.

[00109] As used herein, the terms “nucleic acid,” “nucleic acid molecule,” or “polynucleotide” refer to deoxyribonucleotides or ribonucleotides and polymers thereof in single- or double-stranded form. Unless specifically limited, these terms cover nucleic acids that contain analogues of naturally occurring nucleotides, have binding properties similar to those of the reference nucleic acid, and are metabolized similarly to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleotide sequence, in addition to the explicitly stated sequence, also includes conservatively modified variants (eg, substitutions due to codon degeneracy) and complementary sequences. In particular, substitutions due to codon degeneracy can be obtained by generating sequences in which the third position of one or more of the selected (or all) codons is replaced by mixed base and/or deoxyinosine residues (Batzer et al. (1991) Nucleic Acid Res. 19 :5081; Ohtsuka et al. (1985) J. Biol. Chem. 260:2605-2608; Cassol et al. (1992); Rossolini et al. (1994) Mol. Cell. Probes 8:91-98). The term "nucleic acid" is used interchangeably with the gene, cDNA (complementary DNA), and mRNA (messenger RNA) encoded by the gene. As used herein, the terms “nucleic acid,” “nucleic acid molecule,” or “polynucleotide” are intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), DNA analogues, or RNA produced using nucleotide analogues, and their derivatives, fragments and homologues.

[00110] The term "expression" refers to the biosynthesis of the product encoded by the nucleic acid. For example, in the case of a nucleic acid segment encoding a polypeptide of interest, expression involves transcription of the nucleic acid segment into mRNA and translation of the mRNA into one or more polypeptides.

[00111] The terms “expression unit” and “expression cassette” are used interchangeably herein to refer to a nucleic acid segment encoding a polypeptide of interest and capable of causing expression of the nucleic acid segment in a host cell. The expression unit typically contains a transcription promoter, an open reading frame encoding the polypeptide of interest, and a transcription terminator, all of which are in an operative configuration. In addition to the promoter and transcription terminator, the expression unit may further include other nucleic acid segments, such as, for example, an enhancer or a polyadenylation signal.

[00112] As used herein, the term “expression vector” refers to a nucleic acid molecule, linear or cyclic, containing one or more expression units. In addition to one or more expression units, the expression vector may also include additional nucleic acid segments, such as, for example, one or more origins of replication or one or more selectable markers. Expression vectors are usually derived from plasmid or viral DNA, or they may contain elements of both.

[00113] As used herein, the term "polypeptide", "polypeptide chain" or "protein" refers to a single linear and continuous arrangement of covalently linked amino acids. Polypeptides can form one or more intrachain disulfide bonds. With respect to polypeptides as described herein, reference to modifications or changes to amino acid residues corresponding to those set forth in SEQ ID NO: includes post-translational modifications of such residues. The terms “polypeptide” and “protein” also encompass embodiments where two polypeptide chains are linked to each other in a non-linear manner, for example, through an interchain disulfide bond. For example, a native immunoglobulin molecule consists of two heavy chain polypeptides and two light chain polypeptides. Each of the heavy chain polypeptides binds to a light chain polypeptide through interchain disulfide bonds between the heavy and light chain polypeptides to form two heterodimeric proteins or polypeptides (ie, a protein consisting of two heterologous polypeptide chains). The two heterodimeric proteins then link through additional interchain disulfide bonds between heavy chain polypeptides to form an immunoglobulin protein or polypeptide. As used herein, the protein or polypeptide may be an antibody or an antigen binding fragment of an antibody. In some embodiments, the protein may be a scFv-Fc-scFv protein, a scFv-scFv dimer, or a diabody.

[00114] As will be appreciated by one skilled in the art, proteins and polypeptides are defined herein by the amino acid sequences of individual polypeptide chains, which are designated by reference to SEQ ID NO: throughout this specification. For example, in some embodiments, the scFv-Fc-scFv protein or polypeptide described herein consists of two scFv-Fc-scFv polypeptide chains linked by interchain bonds (e.g., interchain disulfide bonds) to form a dimeric scFv-Fc-scFv protein (eg, homodimeric or heterodimeric protein scFv-Fc-scFv) (see, for example, FIG. 10). In such embodiments, the scFv-Fc-scFv protein is defined by the amino acid sequences of the individual scFv-Fc-scFv polypeptide chains. Polypeptides and proteins may also contain non-peptide components such as carbohydrate groups. Carbohydrates and other non-peptide substituents may be added to a protein or polypeptide by the cell in which the protein is produced, and their composition will vary depending on the type of cell. Proteins and polypeptides are defined herein by the structure of their amino acid backbone; substituents such as carbohydrate groups are usually not specified but may nevertheless be present.

[00115] The terms “amino-terminal” and “carboxy-terminal” are used herein to refer to positions in polypeptides. Where context permits, these terms are used with reference to a specific sequence or portion of a polypeptide to indicate proximity or relative position. For example, a single sequence that is carboxy-terminal to a reference sequence in a polypeptide is located near the carboxy-terminus of said reference sequence, but is not necessarily located at the carboxy-terminus of the entire polypeptide sequence.

[00116] As used herein, the terms “transformation,” “transfection,” and “transduction” refer to the transfer of a nucleic acid (ie, a nucleotide polymer) into a cell. As used herein, the term "genetic transformation" refers to the transfer and incorporation of DNA, particularly recombinant DNA, into a cell. The transferred nucleic acid can be introduced into a cell via an expression vector.

[00117] “Antibody-dependent cell-mediated cytotoxicity” and “ADCC” as used herein refer to a cell-mediated process in which nonspecific cytotoxic cells expressing FcγR (e.g., monocytic cells such as natural killer (NK) cells and macrophages) recognize bound antibody (or other protein capable of binding FcγR) on a target cell and then cause lysis of said target cell. In principle, any effector cell with an activating FcγR can be stimulated to mediate ADCC. The primary cells to mediate ADCC are NK cells, which express only FcγRIII, whereas monocytes, depending on their state of activation, localization, or differentiation, can express FcγRI, FcγRII, and FcγRIII. For a review of FcγR expression on hematopoietic cells, see, for example, Ravetch et al., 1991, Annu. Rev. Immunol., 9:457-92.

[00118] The term “having ADCC activity” as used herein in relation to a polypeptide or protein means that the polypeptide or protein, for example, containing an Fc domain (for example, an immunoglobulin hinge region and an immunoglobulin constant region having CH2 and CH3 domains ), such as those derived from IgG (e.g., IgG1), are capable of mediating antibody-dependent cell-mediated cytotoxicity (ADCC) through binding of a cytolytic Fc receptor (e.g., FcγRIII) on a cytolytic immune effector cell expressing the Fc receptor (e.g., NK cell ). In some embodiments, a polyspecific polypeptide or protein (e.g., a 5T4 x 4-1BB molecule as described herein) containing an Fc domain may have no effector function (e.g., have zero ADCC activity) as a result of mutations in the CH2 domain and/or CH3.

[00119] “Complement-dependent cytotoxicity” and “CDC” as used herein refer to the process in which components in normal serum (“complement”), together with an antibody or other C1q complement-fixing protein bound to a target antigen, cause cell lysis. target expressing the target antigen. Complement consists of a group of serum proteins that act in concert and in an orderly sequence to produce their effects.

[00120] The terms “classical complement pathway” and “classical complement system” as used herein are synonymous and refer to a specific pathway of complement activation. The classical pathway requires antigen-antibody complexes for initiation and involves the ordered activation of nine major component proteins, designated C1-C9. At several stages of the activation process, the product is an enzyme that catalyzes the subsequent stage. This cascade enables the amplification and activation of large amounts of complement from a relatively small input signal.

[00121] The term “having CDC activity” as used herein in relation to a polypeptide or protein means that the polypeptide or protein, for example, containing an Fc domain (for example, an immunoglobulin hinge region and an immunoglobulin constant region having CH2 and CH3 domains ), such as those derived from IgG (eg, IgG1), are capable of mediating complement-dependent cytotoxicity (CDC) by binding complement protein C1q and activating the classical complement system. In some embodiments, a polyspecific polypeptide or protein (e.g., a 5T4 x 4-1BB molecule as described herein) may have no effector function (e.g., have zero CDC activity) as a result of one or more mutations in the CH2 and/or domains CH3.

[00122] “Enhanced effector cell activation” as used herein refers to enhancing, prolonging and/or enhancing the effector cell response to the polypeptides or proteins described herein. In some embodiments, increased activation of effector cells refers to increased cytotoxic activity of the effector cell. In some embodiments, enhanced effector cell activation refers to increased cytokine production, cell proliferation, or altered cell surface expression of a molecule such that the effector cell's ability to lyse a target cell is enhanced. In certain embodiments, the polypeptides and proteins described herein enhance effector cell activation by modulating signaling through the Wnt/β-catenin pathway.

[00123] As used herein, the term “effector cell” refers to an immune system cell capable of lysing or destroying a target cell, such as a tumor cell. As used herein, an effector cell may refer to a lymphocyte such as a T cell, natural killer (NK) or NKT cell, monocyte, macrophage, dendritic cell, or granulocyte. In particular embodiments, the term “effector cell” refers to a T cell, NK cell, or NKT cell.

[00124] As used herein, the terms “treatment” or “tapering” refer to either therapeutic treatment or prophylactic/prophylactic treatment. Treatment is therapeutic if at least one symptom of the disease in the individual receiving treatment improves, or the treatment can delay the worsening of the individual's progressive disease or prevent the occurrence of additional comorbidities.

[00125] As used herein, the term “therapeutically effective amount (or dose)” or “effective amount (or dose)” of a polypeptide or protein described herein, or a composition containing the same, refers to that amount of a compound that is sufficient to reduce the intensity of one or more symptoms of a disease being treated in a statistically significant manner, or a statistically significant improvement in organ function. When applied to a single active ingredient administered separately, the therapeutically effective dose refers to that ingredient alone. In the context of a combination, a therapeutically effective dose refers to the combined amounts of active ingredients that produce a therapeutic effect, whether administered sequentially or simultaneously (in the same formulation or simultaneously in separate formulations).

[00126] As used herein, the term “statistical significance” refers to the probability of obtaining a test result that occurs by chance. For example, an observation or test result is considered statistically significant if the probability of it occurring by chance (p) is less than a specified statistical threshold (α), or p < α. The statistical threshold for a particular test can be set in accordance with data characteristics and rules known in the art. For example, α is typically set at 5% (0.05), so that p < 0.05 must be met for an individual result to be considered statistically significant.

[00127] As used herein, “5T4-binding domain” refers to a domain of a polypeptide described herein that specifically binds oncofetal trophoblastic glycoprotein (5T4) (e.g., human 5T4), also known as TPBG and Wnt-activated inhibitory factor 1 (WAIF1). 5T4 is a 72 kDa oncofetal glycoprotein that is highly N-glycosylated proteins with several leucine-rich repeats linked by protein-protein interactions. The term "5T4" can refer to any isoform of 5T4. Exemplary human 5T4 nucleotide sequences are provided in Table 1 below as well as SEQ ID NOs: 163 and 167, and exemplary human 5T4 amino acid sequences are provided in SEQ ID NOs: 164 and 168.

[00128] As used herein, “4-1BB binding domain” refers to the binding domain of a protein or polypeptide described herein that is capable of specifically binding to human 4-1BB (also known as CD137). The term "4-1BB" can refer to any isoform of 4-1BB. Exemplary nucleotide sequences of human 4-1BB are presented in Table 1 below as well as SEQ ID NOs: 161 and 165, and exemplary amino acid sequences of human 4-1BB are presented in SEQ ID NOs: 162 and 166.

Table 1. Exemplary DNA sequences and amino acid sequences of 4-1BB and 5T4

Protein name DNA sequence SEQ ID NO: DNA sequences Amino acid sequence SEQ ID NO: amino acid sequence Full size human 4-1BB ATGGGAAACAGCTGTTACAACATAGTAGCCACTCTGTTGCTGGTCCTCAACTTTGAGAGGACAAGATCATTGCAGGATCCTTGTAGTAACTGCCCAGCTGGTACATTCTGTGATAATAACAGGAATCAGATTTGCAGTCCCTGTCCTCCAAATAGTTTCTCCAGCGCAGGTGGACAAAGGACCTGTGACATATGCAGGCAGTGTAAAGGTGTTTTCAGGACCAGGAAGGAGTGTTCCTCCACC AGCAATGCAGAGTGTGACTGCACTCCAGGGTTTCACTGCCTGGGGGCAGGATGCAGCATGTGTGAACAGGATTGTAAACAAGGTCAAGAACTGACAAAAAAAGGTTGTAAAGACTGTTGCTTTGGGACATTTAACGATCAGAAACGTGGCATCTGTCGACCCTGGACAAACTGTTCTTTGGATGGAAAGTCTGTGCTTGTGAATGGGACGAAGGAGAGGGACGTGGTCTGTGGACCATCTCCAGCC GACCTCTCTCCGGGAGCATCCTCTGTGACCCCGCCTGCCCCTGCGAGAGAGCCAGGACACTCTCCGCAGATCATCTCCTTCTTTCTTGCGCTGACGTCGACTGCGTTGCTCTTCCTGCTGTTCTTCCTCACGCTCCGTTTCTCTGTTGTTAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTC CAGAAGAAGAAGAAGGAGGATGTGAACTGTGA 161 MGNSCYNIVATLLLLVLNFERTRSLQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPPADSPGASSVTPPAPAREPGHSPQIISFFLALTSTALLFLLFFLTLRFSV VKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL 162 Full size human 5T4 ATGCCTGGGGGGTGCTCCCGGGGCCCCGCCGCCGGGGACGGGCGTCTGCGGCTGGCGCGACTAGCGCTGGTACTCCTGGGCTGGGTCTCCTCGTCTTCTCCCACCTCCTCGCATCCTCCTTCTCCTCCTCGGCGCCGTTCCTGGCTTCCGCCGTGTCCGCCCAGCCCCGCTGCCGGACCAGTGCCCCGCGCTGTGCGAGTGCTCCGAGGCAGCGCGCACAGTCAAGTGCGTTAACCG CAATCTGACCGAGGTGCCCACGGACCTGCCCGCCTACGTGCGCAACCTCTTCCTTACCGGCAACCAGCTGGCCGTGCTCCCTGCCGGCGCCTTCGCCCGCCGGCCGCCGCTGGCGGAGCTGGCCGCGCTCAACCTCAGCGGCAGCCGCCTGGACGAGGTGCGCGCGGGCGCCTTCGAGCATCTGCCCAGCCTGCGCCAGCTCGACCTCAGCCACAACCCACTGGCCGACCTCAGTCCCTTCGCTTTCT CGGGCAGCAATGCCACGTCTCGGCCCCCAGTCCCCTTGTGGAACTGATCCTGAACCACATCGTGCCCCCTGAAGATGAGCGGCAGAACCGGAGCTTCGAGGGCATGGTGGTGGCGGCCCTGCTGGCGGGCCGTGCACTGCAGGGGCTCCGCCGCTTGGAGCTGGCCAGCAACCACTTCCTTTACCTGCCGCGGGATGTGCTGGCCCAACTGCCCAGCCTCAGGCACCTGGACTTAAGTAATAATTCG CTGGTGATGAGCCTGACCTACGTGTCCTTCCGCAACCTGACACATCTAGAAAGCCTCCACCTGGAGGACAATGCCCTCAAGGTCCTTCACAATGGCACCCTGGCTGAGTTGCAAGGTCTACCCCACATTAGGGTTTTCCTGGACAACAATCCCTGGGTCTGCGACTGCCACATGGCAGACATGGTGACCTGGCTCAAGGAAACAGAGGTAGTGCAGGGCAAAGACCGGCTCACCTGTGCATATCCGGAAAAAATG AGGAATCGGGTCCTCTTGGAACTCAACAGTGCTGACCTGGACTGTGACCCGATTCTTCCCCCATCCCTGCAAACCTCTTATGTCTTCCTGGGTATTGTTTTAGCCCTGATAGGCGCTATTTTCCTCCTGGTTTTGTATTTGAACCGCAAGGGGATAAAAAAGTGGATGCATAACATCAGAGATGCCTGCAGGGATCACATGGAAGGGTATCATTACAGATATGAAATCAATGCGGACCCCAGATTA ACGAACCTCAGTTCTAACTCGGATGTCTGA 163 MPGGCSRGPAAGDGRLRLARLALVLLGWVSSSSPTSSASSFSSSAPFLASAVSAQPPLPDQCPALCECSEAARTVKCVNRNLTEVPTDLPAYVRNLFLTGNQLAVLPAGAFARRPPLAELAALNLSGSRLDEVRAGAFEHLPSLRQLDLSHNPLADLSPFAFSGSNASVSAPSPLVELILNHIVPPEDERQNRSFEGMVVAALLAGRALQGLRRLELASNHFLYLP RDVLAQLPSLRHLDLSNNSLVSLTYVSFRNLTHLESLHLEDNALKVLHNGTLAELQGLPHIRVFLDNNPWVCDCHMADMVTWLKETEVVQGKDRLTCAYPEKMRNRVLLELNSADLDCDPILPPSLQTSYVFLGIVLALIGAIFLLVLYLNRKGIKKWMHNIRDACRDHMEGYHYRYEINADPRLTNLSSNSDV 164 human 4-1BB (ECD*) ATGGGAAACAGCTGTTACAACATAGTAGCCACTCTGTTGCTGGTCCTCAACTTTGAGAGGACAAGATCATTGCAGGATCCTTGTAGTAACTGCCCAGCTGGTACATTCTGTGATAATAACAGGAATCAGATTTGCAGTCCCTGTCCTCCAAATAGTTTCTCCAGCGCAGGTGGACAAAGGACCTGTGACATATGCAGGCAGTGTAAAGGTGTTTTCAGGACCAGGAAGGAGTGTTCCTCCACC AGCAATGCAGAGTGTGACTGCACTCCAGGGTTTCACTGCCTGGGGGCAGGATGCAGCATGTGTGAACAGGATTGTAAACAAGGTCAAGAACTGACAAAAAAAGGTTGTAAAGACTGTTGCTTTGGGACATTTAACGATCAGAAACGTGGCATCTGTCGACCCTGGACAAACTGTTCTTTGGATGGAAAGTCTGTGCTTGTGAATGGGACGAAGGAGAGGGACGTGGTCTGTGGACCATCTCCAGCC GACCTCTCTCCGGGAGCATCCTCTGTGACCCCGCCTGCCCCTGCGAGAGAGCCAGGACACTCTCCGCAG 165 MGNSCYNIVATLLLLVLNFERTRSLQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQ 166 human 5T4 (ECD*) ATGCCTGGGGGGTGCTCCCGGGGCCCCGCCGCCGGGGACGGGCGTCTGCGGCTGGCGCGACTAGCGCTGGTACTCCTGGGCTGGGTCTCCTCGTCTTCTCCCACCTCCTCGCATCCTCCTTCTCCTCCTCGGCGCCGTTCCTGGCTTCCGCCGTGTCCGCCCAGCCCCGCTGCCGGACCAGTGCCCCGCGCTGTGCGAGTGCTCCGAGGCAGCGCGCACAGTCAAGTGCGTTAACCG CAATCTGACCGAGGTGCCCACGGACCTGCCCGCCTACGTGCGCAACCTCTTCCTTACCGGCAACCAGCTGGCCGTGCTCCCTGCCGGCGCCTTCGCCCGCCGGCCGCCGCTGGCGGAGCTGGCCGCGCTCAACCTCAGCGGCAGCCGCCTGGACGAGGTGCGCGCGGGCGCCTTCGAGCATCTGCCCAGCCTGCGCCAGCTCGACCTCAGCCACAACCCACTGGCCGACCTCAGTCCCTTCGCTTTCT CGGGCAGCAATGCCACGTCTCGGCCCCCAGTCCCCTTGTGGAACTGATCCTGAACCACATCGTGCCCCCTGAAGATGAGCGGCAGAACCGGAGCTTCGAGGGCATGGTGGTGGCGGCCCTGCTGGCGGGCCGTGCACTGCAGGGGCTCCGCCGCTTGGAGCTGGCCAGCAACCACTTCCTTTACCTGCCGCGGGATGTGCTGGCCCAACTGCCCAGCCTCAGGCACCTGGACTTAAGTAATAATTCG CTGGTGATGAGCCTGACCTACGTGTCCTTCCGCAACCTGACACATCTAGAAAGCCTCCACCTGGAGGACAATGCCCTCAAGGTCCTTCACAATGGCACCCTGGCTGAGTTGCAAGGTCTACCCCACATTAGGGTTTTCCTGGACAACAATCCCTGGGTCTGCGACTGCCACATGGCAGACATGGTGACCTGGCTCAAGGAAACAGAGGTAGTGCAGGGCAAAGACCGGCTCACCTGTGCATATCCGGAAAAAATG AGGAATCGGGTCCTCTTGGAACTCAACAGTGCTGACCTGGACTGTGACCCGATTCTTCCCCCATCCCTGCAAACCCTT 167 MPGGCSRGPAAGDGRLRLARLALVLLGWVSSSSPTSSASSFSSSAPFLASAVSAQPPLPDQCPALCECSEAARTVKCVNRNLTEVPTDLPAYVRNLFLTGNQLAVLPAGAFARRPPLAELAALNLSGSRLDEVRAGAFEHLPSLRQLDLSHNPLADLSPFAFSGSNASVSAPSPLVELILNHIVPPEDERQNRSFEGMVVAALLAGRALQGLRRLELASNHFLYLP RDVLAQLPSLRHLDLSNNSLVSLTYVSFRNLTHLESLHLEDNALKVLHNGTLAELQGLPHIRVFLDNNPWVCDCHMADMVTWLKETEVVQGKDRLTCAYPEKMRNRVLLELNSADLDCDPILPPSLQTS 168

*ECD = extracellular domain

[00129] As used herein, the term “polyspecific polypeptide” refers to a polypeptide containing two or more binding domains, each of which is capable of specifically binding to a target antigen. For example, the polypeptides described herein may contain 2, 3, 4 or more binding domains and may be capable of binding 2, 3, 4 or more target antigens. In some embodiments, the polyspecific polypeptide is a bispecific polypeptide. As used herein, a “bispecific polypeptide” contains two binding domains and is capable of binding to two different target antigens. In some embodiments, the bispecific polypeptides described herein comprise a first binding domain that specifically binds to a cell surface antigen expressed on a target cell, such as 5T4. In some embodiments, the bispecific polypeptides described herein comprise a binding domain that specifically binds to a cell surface antigen expressed on an effector cell, such as 4-1BB. In particular embodiments, the polyspecific polypeptide is a homodimer of the ADAPTIR™ bispecific polypeptide in the scFv-Fc-scFv format.

[00130] Polyspecific polypeptides using scaffolds are disclosed, for example, in PCT Application Publications Nos. WO 2007/146968; WO 2010/040105; WO 2010/003108; WO 2016/094873; WO 2017/053469; US Patent Application Publication No. 2006/0051844; and US Patent Nos. 7,166,707; and 8,409,577, each of which is incorporated herein by reference in its entirety. In certain embodiments, the polyspecific polypeptides described herein are bispecific polypeptides and may comprise a scFv-Fc-scFv structure, also referred to herein as an ADAPTIR ^™ polypeptide, or format 1, an exemplary embodiment of which is shown in FIG. 10. The structure of a polypeptide containing the structure of format 1 contains, in the direction from the N-terminus to the C-terminus: the first scFv binding domain - the immunoglobulin (Ig) hinge region - the Ig constant region - the second scFv binding domain (FIG. 10) . In certain embodiments, the polyspecific polypeptides described herein may comprise an IgG-scFv structure (also referred to herein as Morrison format or format 2). The structure of the polypeptide containing the structure of format 2 contains, in the direction from the N-terminus to the C-terminus: scFv binding domain - Ig constant region - Ig hinge region - Ig variable region. Format 2 is essentially an intact Ig molecule containing a C-terminal scFv domain.

Binding polypeptides and proteins

[00131] In some embodiments, the present invention provides polypeptides capable of specifically binding to 5T4 (eg, 5T4-binding polypeptides), as well as polyspecific polypeptides and proteins containing these binding domains. Such embodiments may be referred to as 5T4 binding polypeptides. In particular embodiments, the present invention relates to multispecific binding proteins comprising a 5T4 binding domain and a second binding domain capable of binding to a cell surface molecule on an effector cell. In particular embodiments, the present invention provides bispecific binding proteins comprising a 5T4 binding domain and a second binding domain capable of binding to 4-1BB (eg, a 4-1BB binding domain). In some embodiments, the 5T4-binding polypeptides comprise the following structure, from N-terminus to C-terminus: 5T4-binding domain - Fc domain.

[00132] In some embodiments, the present invention provides polypeptides capable of specifically binding to 4-1BB, as well as polyspecific polypeptides and proteins containing these binding domains. Such embodiments may be referred to as 4-1BB binding polypeptides. In particular embodiments, the present invention relates to polyspecific polypeptides comprising a 4-1BB binding domain and a second binding domain capable of binding to a cell surface molecule on a target cell. In particular embodiments, the present invention provides bispecific polypeptides comprising a 4-1BB binding domain and a second binding domain capable of binding to 5T4 (eg, a 5T4 binding domain). In some embodiments, the 4-1BB binding polypeptides comprise the following structure, from N-terminus to C-terminus: 4-1BB binding domain - Fc domain.

[00133] In some embodiments, the polypeptides described herein may further comprise immunoglobulin constant regions, linker peptides, hinge regions, immunoglobulin dimerization/heterodimerization domains, connecting amino acids, tags, etc. These components of the disclosed polypeptides and proteins are described in more detail. are described below.

[00134] The polypeptides and proteins described herein (eg, 5T4-binding polypeptides, 4-1BB-binding polypeptides and/or polyspecific binding proteins containing them) contain one or more binding domains capable of specifically binding a target antigen. The binding domains described herein can be in the form of an antibody or fusion protein in any of a variety of different formats (eg, the fusion protein can be in the form of a bispecific or multispecific molecule). Non-limiting examples of bispecific molecules include a scFv-Fc-scFv molecule (eg, a bispecific protein containing a format 1 structure), a scFv-Ig molecule (eg, a bispecific protein containing a format 2 structure), and a scFv-scFv molecule. In some embodiments, the bispecific molecules described herein comprise or consist of a first scFv binding domain linked to a second scFv binding domain, and do not include other sequences such as an immunoglobulin constant region. In other embodiments, the bispecific protein described herein is a diabody.

[00135] In some embodiments, the polypeptides and proteins described herein are conjugated to a drug or toxic moiety.

[00136] In some embodiments, the polypeptides and proteins described herein (e.g., 5T4 binding polypeptides, 4-1BB binding polypeptides, and/or polyspecific binding proteins containing them) may comprise the amino acid and/or nucleic acid sequences set forth in tables 2-10. In certain embodiments, the binding domains of the polypeptides described herein comprise (i) an immunoglobulin light chain variable region ( _VL ) comprising the CDRs LCDR1, LCDR2, and LCDR3, and (ii) an immunoglobulin heavy chain variable region ( _VH ) comprising CDR HCDR1, HCDR2 and HCDR3. In some embodiments, the amino acid sequences provided for the polypeptide constructs do not include human immunoglobulin leader sequences. The CDR sequences and amino acid substitution positions shown are determined according to IMGT criteria (Brochet et al, Nucl. Acids Res. (2008) 36, W503-508). In some cases, the sequences shown in the description, including in tables 2-10, contain amino acid substitutions relative to the original sequence (for example, antibodies to 4-1BB or its binding fragment, such as clone 1618/1619 disclosed in PCT application publication no. WO 2016/185016, or an antibody to 5T4 or a binding fragment thereof, such as disclosed in PCT Application Publication No. WO 2016/185016). In some embodiments, the original 5T4 binding domain sequence comprises the amino acid sequence set forth in SEQ ID NOs: 38 and 68. In some embodiments, the original 4-1BB binding domain sequence comprises the amino acid sequence set forth in SEQ ID NOs: 28 and 16. Exemplary parent sequences of the 4-1BB and 5T4 binding domains described herein are presented below in Table 2. Underlined text denotes CDR sequences.

Table 2. Exemplary starting sequences of binding domains

Original sequence Amino acid sequence SEQ ID NO: V _H 1618 to 4-1BB EVQLLESGGGLVQPGGSLRLSCAAS GFTFSYGS MYWVRQAPGKGLEWVSS ISSGSGST YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC ARSSYYGSYYSIDY WGQGTLVTVSS 28 V _L 1618 k 4-1BB DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYDNLPTFGQGTKLEIK 16 _VH 1210 to 5T4 EVQLLESGGGLVQPGGSLRLSCAAS GFTFSSYA MSWVRQAPGKGLEWVSA ISGSGGST YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC ARYYGGYYSAWMDY WGQGTLVTVSS 38 V _L 1210 to 5T4 DIQMTQSPSSLSSASVGDRVTITCRAS QSISSY LNWYQQKPGKAPKLLIY AAS SLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQTYGYLHT FGQGTKLEIK 68

[00137] In certain embodiments, the V _L and/or V _H region of the binding domain of the present invention is derived from the V _L and/or V _H region of the original V _L and/or V _H region (e.g., 1618/1619, as described in the application publication PCT No. WO 2016/185016) and optionally contains about one or more (for example, about 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, about one or more (for example, about 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, about one or more (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conservative amino acid substitutions or non-conservative amino acid substitutions), or a combination of the above changes compared to the V _L and/or V _H sequence of a known monoclonal antibody. The insertion(s), deletion(s) or substitution(s) may occur anywhere in the V _L and/or V _H region, including at the amino or carboxy terminus, or at both ends of this region, provided that each The CDR contains zero changes or no more than one, two, or three changes. In some embodiments, the binding domain comprising a modified V _L and/or V _H region retains the ability to specifically bind its target with an affinity similar to or greater than that of the original binding domain.

[00138] In some embodiments, the binding domain described herein is derived from an antibody and comprises a variable heavy chain ( _VH ) and a variable light chain (VL ₎ . For example, scFv containing V _H and V _L chains. These binding domains and variable chains can be arranged in any order that maintains some degree of binding to the target(s). For example, the variable domains may be arranged in the following order: V _H 5T4 - V _L 5T4 - V _H 4-1BB - V _L 4-1BB; V _L 5T4 - V _H 5T4 - V _H 4-1BB - V _L 4-1BB; V _H 5T4 - V _L 5T4 - V _L 4-1BB - V _H 4-1BB; V _L 5T4 - V _H 5T4 - V _L 4-1BB - V _H 4-1BB; V _H 4-1BB - V _L 4-1BB - V _H 5T4 - V _L 5T4; V _L 4-1BB - V _H 4-1BB - V _L 5T4 - V _H 5T4; V _H 4-1BB - V _L 4-1BB - V _L 5T4 - V _H 5T4; or V _L 4-1BB - V _H 4-1BB - V _H 5T4 - V _L 5T4. Pairs of V _H regions and V _L regions in the 4-1BB binding domain and/or the 5T4 binding domain may be in a single chain antibody (scFv) format.

[00139] In some embodiments, the polypeptides and proteins described herein (eg, 5T4-binding polypeptides, 4-1BB-binding polypeptides, and/or polyspecific binding proteins containing them) contain scFv binding domains. In such embodiments, the binding domains may be referred to as scFv domains. In some embodiments, the binding domain is a single-chain Fv fragment (scFv) containing V _H and V _L regions specific to the target of interest. In certain embodiments, the V _H and V _L regions are human or humanized. In some variations, the binding domain is a single chain Fv (scFv) containing V _L and V _H regions connected by a peptide linker.

[00140] The use of peptide linkers to connect the V _L and V _H regions is well known in the art, and a large number of publications exist in this particular field. In some embodiments, the peptide linker is 15-mer and consists of three repeats of the amino acid sequence Gly-Gly-Gly-Gly-Ser ((Gly ₄ Ser) ₃ ) (SEQ ID NO: 96). Other linkers have been used, and phage display technology as well as selectively infecting phage technology have been used to diversify and select suitable linker sequences (Tang et al. , J. Biol. Chem. 271, 15682-15686, 1996; Hennecke et al. , Protein Eng. 11, 405-410, 1998). In certain embodiments, the V _L and V _H regions are connected by a peptide linker having an amino acid sequence containing the structure of the formula (Gly ₄ Ser)n, where n = 1-5. For example, in one embodiment, the linker contains (Gly4Ser) ₄ . Other suitable linkers can be obtained by optimizing a simple linker (eg, (Gly ₄ Ser) _n ) through random mutagenesis. In some embodiments, the V _H region of the scFv described herein may be located N-terminal to the linker sequence. In some embodiments, the V _L region of the scFv described herein may be located C-terminal to the linker sequence. In some embodiments, the scFv may bind to 5T4 and/or 4-1BB more efficiently than an antibody containing the same V _H and V _L region sequences in the same orientation. In certain embodiments, scFv may bind more efficiently to 5T4 and/or 4-1BB in the V _L -V _H orientation compared to the V _H -V _L orientation, or vice versa.

[00141] In some embodiments, the polypeptide comprises a binding domain linker connecting binding domains (eg, connecting scFv domains). In some embodiments, the binding domain linker is a Gly ₄ Ser linker. In some embodiments, the binding domain linker is 20-mer and consists of four repeats of the amino acid sequence Gly-Gly-Gly-Gly-Ser ((Gly ₄ Ser) ₃ ) (SEQ ID NO: 98). In some embodiments, the binding domain linker comprises an amino acid sequence selected from SEQ ID NO: 86-108. Other linkers have been used, and phage display technology as well as selectively infecting phage technology have been used to diversify and select suitable linker sequences (Tang et al. , J. Biol. Chem. 271, 15682-15686, 1996; Hennecke et al. , Protein Eng. 11, 405-410, 1998). In certain embodiments, the V _L and V _H regions are connected by a peptide linker having an amino acid sequence containing the structure of the formula (Gly ₄ Ser) _n , where n = 1-5. Other suitable linkers can be obtained by optimizing a simple linker (eg, (Gly ₄ Ser) _n ) through random mutagenesis. In some cases, the bispecific molecule may contain a 5T4-binding scFv linked to a 4-1BB-binding scFv. In some embodiments, the bispecific molecules do not contain a hinge region or constant region.

[00142] In some embodiments, the polypeptides and proteins described herein (eg, 5T4 binding polypeptides, 4-1BB binding polypeptides, and/or polyspecific binding proteins containing them) further comprise a hinge. In some embodiments, the hinge is a modified immunoglobulin hinge in which one or more cysteine residues in the hinge region of the wild-type immunoglobulin are replaced by one or more other amino acid residues (eg, serine or alanine). Exemplary modified immunoglobulin hinges, carboxy-terminal linkers, and amino-terminal linkers include a human immunoglobulin IgG1 hinge region in which one, two, or three cysteine residues contained in the wild-type human IgG1 hinge are replaced with one, two, or three different amino acid residues (e.g., serine or alanine ). The altered immunoglobulin hinge may further comprise replacing the proline with another amino acid (eg, serine or alanine). For example, the altered human IgG1 hinge described above may further comprise replacing the proline located carboxy-terminal to the three cysteines of the wild-type human IgG1 hinge region with another amino acid residue (eg, serine, alanine). In one embodiment, the prolines of the core hinge region are not replaced. In certain embodiments, the hinge, carboxy-terminal linker, or amino-terminal linker polypeptide contains or is at least 80%, at least 81%, at least 82%, at least 83%, 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a wild-type immunoglobulin hinge region, such as a wild-type human IgG1 hinge, a wild-type human IgG2 hinge, or a wild-type human IgG4 hinge.

[00143] In certain embodiments, a hinge present in a polypeptide that forms a heterodimer with another polypeptide chain may be an immunoglobulin hinge, such as a wild-type immunoglobulin hinge region or a modified hinge region thereof. In certain embodiments, the hinge of one polypeptide chain of a heterodimeric protein is identical to the corresponding hinge of another polypeptide chain of the heterodimer. In some other embodiments, the hinge of one chain is different from the hinge of the other chain (in length or sequence). Different hinges in different chains allow different effects on the binding affinities of the binding domains to which the hinges are associated, such that the heterodimer is able to selectively bind to the target of one binding domain over the target of another binding domain. For example, in certain embodiments, the heterodimeric protein has a 4-1BB binding domain on one strand and a 5T4 binding domain on the other strand. The presence of two different hinges in the two chains may allow the heterodimer to bind first to 5T4 and then to 4-1BB. Thus, the heterodimer can recruit 4-1BB-expressing effector cells to 5T4-expressing target cells (eg, 5T4-expressing tumor or cancer cells), which in turn can damage or kill the 5T4-expressing cells.

[00144] In certain embodiments, the carboxy-terminal linker or amino-terminal linker is a flexible linker sequence containing glycine-serine repeats (eg, Gly ₄ Ser). In certain embodiments, the linker contains three Gly ₄ Ser repeats (SEQ ID NO: 96) followed by a proline residue. In certain embodiments, the proline residue is followed by an amino acid selected from the group consisting of glycine, arginine, and serine. In some embodiments, the carboxy-terminal linker or amino-terminal linker comprises or consists of a sequence selected from SEQ ID NOs: 86 - 108.

[00145] Some exemplary hinge, carboxy-terminal linker and amino-terminal linker sequences suitable for use in accordance with the present invention are presented below in Table 3. Additional exemplary hinge and linker regions are presented in SEQ ID NOs: 241-244, 601, 78, 763 -791, 228, 379-434, 618-749 contained in US Patent Publication No. 2013/0129723 (these sequences are incorporated herein by reference).

Table 3. Exemplary loops and linkers

Hinge area Amino acid sequence SEQ ID NO: hinge sss(s)-hIgG1 EPKSSDKTHTSPPSS 71 hinge csc(s)-hIgG1 EPKSCDKTHTSPPCS 72 hinge ssc(s)-hIgG1 EPKSSDKTHTSPPCS 73 hinge scc(s)-hIgG1 EPKSSDKTHTCPPCS 74 hinge css(s)-hIgG1 EPKSCDKTHTSPPSS 75 hinge scs(s)-hIgG1 EPKSSDKTHTCPPSS 76 hinge ccc(s)-hIgG1 EPKSCDKTHTSPPCS 77 hinge ccc(p)-hIgG1 EPKSCDKTHTSPPCP 78 hinge sss(p)-hIgG1 EPKSSDKTHTSPPSP 79 hinge csc(p)-hIgG1 EPKSCDKTHTSPPCP 80 hinge ssc(p)-hIgG1 EPKSSDKTHTSPPCP 81 hinge scc(p)-hIgG1 EPKSSDKTHTCPPCP 82 hinge css(p)-hIgG1 EPKSCDKTHTSPPSP 83 hinge scs(p)-hIgG1 EPKSSDKTHTCPPSP 84 Scppcp SCPPCP 85 STD1 NYGGGGSGGGGSGGGGSGNS 86 STD2 NYGGGGSGGGGSGGGGSGNYGGGGSGGGGSGGGGSGNS 87 H1 N.S. 88 H2 GGGGSGNS 89 H3 NYGGGGSGNS 90 H4 GGGGSGGGGSGNS 91 H5 NYGGGGSGGGGGSGNS 92 H6 GGGGSGGGGSGGGGSGNS 93 H7 GCPPCPNS 94 Gly ₄ Ser GGGGS 95 (G ₄ S) ₃ GGGGSGGGGSGGGGS 96 H105 SGGGGSGGGGSGGGGS 97 (G ₄ S) ₄ GGGGSGGGGSGGGGSGGGGS 98 H75 (NKG2A quadruple mutant) QRHNNSSLNTGTQMAGHSPNS 99 H83 (derived from NKG2A) SSLNTGTQMAGHSPNS 100 H106 (derived from NKG2A) QRHNNSSLNTGTQMAGHS 101 H81 (derived from NKG2D) EVQIPLTESYSPNS 102 H91 (derived from NKG2D) NSLANQEVQIPLTESYSPNS 103 H94 SGGGGSGGGGSGGGGSPNS 104 H111 SGGGGSGGGGSGGGGSPGS 105 H113 SGGGGSGGGGSGGGGSPAS 106 H114 SGGGGSGGGGSGGGGSPS 107 H115 SGGGGSGGGGSGGGGSPSS 108

[00146] In other embodiments, the polypeptides and proteins described herein include a heterodimerization domain capable of heterodimerizing with another heterodimerization domain on a second, non-identical polypeptide chain. In certain variations, the second polypeptide chain for heterodimerization includes a second binding domain. Accordingly, in certain embodiments of the present invention, two non-identical polypeptide chains, one of which contains a polypeptide containing a first binding domain and the second optionally contains a second binding domain, dimerize to form a heterodimeric binding protein. Dimerization/heterodimerization domains can be used in cases where the formation of heterodimers from two non-identical polypeptide chains is desired, where one or both polypeptide chains contain a binding domain. In certain embodiments, one polypeptide chain member of the individual heterodimers described herein does not contain a binding domain. Examples of heterodimer types include those described in US Patent Application Publications Nos. 2013/0095097 and 2013/0129723, and PCT International Application Publication No. WO 2016/094873.

[00147] In certain embodiments, the first and second polypeptide chains are dimerized by including an “immunoglobulin dimerization domain” or an “immunoglobulin heterodimerization domain.” The term "immunoglobulin dimerization domain" or "immunoglobulin heterodimerization domain" as used herein refers to an immunoglobulin domain of a first polypeptide chain that selectively interacts with or binds to another immunoglobulin domain of a second polypeptide chain, wherein the interaction of the different immunoglobulin domains contributes significantly to the heterodimerization of the first and second polypeptide chains. chains (i.e., the formation of a dimer between two different polypeptide chains, also called a “heterodimer”), or effectively stimulates it. The immunoglobulin heterodimerization domains in the heterodimer polypeptide chains are distinct from each other and thus can be selectively modified to promote heterodimerization of both chains and minimize homodimerization of either chain. The immunoglobulin heterodimerization domains provided herein provide efficient heterodimerization between different polypeptides and facilitate purification of the resulting heterodimeric protein.

[00148] As described herein, immunoglobulin heterodimerization domains useful for promoting heterodimerization of two different polypeptide chains according to the present invention include immunoglobulin CH1 and CL domains, both wild type and altered, for example, human immunoglobulin CH1 and CL domains. In certain embodiments, the immunoglobulin heterodimerization domain is a wild-type CH1 domain, such as a wild-type IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, or IgM CH1 domain, for example, as set forth in SEQ ID NO: 114, 186 -192 and 194, respectively, from US Patent Application Publication No. 2013/0129723, or SEQ ID NO: 114 from US Patent Application Publication No. 2013/0129723 (the said sequence is incorporated herein by reference). In additional embodiments, a cysteine residue of a wild-type CH1 domain (e.g., human CH1) involved in forming a disulfide bond with the CL domain of a wild-type immunoglobulin (e.g., human CL) is removed or replaced in the altered CH1 domain of the immunoglobulin such that the disulfide bond between the altered is not formed by the wild-type CH1 domain and the CL domain.

[00149] In certain embodiments, the immunoglobulin heterodimerization domain is a wild-type CL domain, such as a wild-type Cκ domain or a wild-type Cλ domain, for example, as set forth in SEQ ID NOs: 112 and 113, respectively, from U.S. Patent Application Publication No. 2013/0129723 (these sequences are incorporated herein by reference). In further embodiments, the immunoglobulin heterodimerization domain is an altered CL domain of an immunoglobulin, such as an altered Cκ or Cλ domain, for example, an altered Cκ or Cλ domain of a human immunoglobulin. In certain embodiments, a cysteine residue in the wild-type CL domain involved in the formation of a disulfide bond with the CH1 domain of the wild-type immunoglobulin is removed or replaced in an altered CL domain of the immunoglobulin, e.g., in the Cκ domain, as set forth in SEQ ID NO: 141 of the patent publication US Application No. 2013/0129723, or the Cλ domain as set forth in SEQ ID NO: 140 of US Patent Application Publication No. 2013/0129723 (these sequences are incorporated herein by reference). In certain embodiments, only the last cysteine of the wild-type human Cκ domain is removed from the altered Cκ domain, since the first arginine removed from the wild-type human Cκ domain can be provided by a linker containing an arginine at its carboxy terminus and linking the amino terminus of the altered Cκ domain to another domain (eg, an immunoglobulin subregion, such as the subregion containing the immunoglobulin CH2 and CH3 domains).

[00150] In further embodiments, the immunoglobulin heterodimerization domain is an altered Cκ domain containing one or more amino acid substitutions relative to the wild-type Cκ domain at positions that may participate in the formation of an interchain hydrogen bonding network at the Cκ-Cκ interface. For example, in certain embodiments, the immunoglobulin heterodimerization domain is a modified human Cκ domain in which one or more amino acids at positions N29, N30, Q52, V55, T56, S68, or T70 are replaced with another amino acid. Amino acid numbering is based on their positions in the modified human Cκ sequence set forth in SEQ ID NO: 141 from US Patent Application Publication No. 2013/0129723 (the said sequence is incorporated herein by reference). In certain embodiments, the immunoglobulin heterodimerization domain is a modified human Cκ domain having one, two, three, or four amino acid substitutions at positions N29, N30, V55, or T70. The amino acid used as a substitute at the above positions may be alanine or an amino acid residue with a bulky side chain moiety such as arginine, tryptophan, tyrosine, glutamate, glutamine, lysine, aspartate, methionine, serine or phenylalanine. Altered human Cκ domains are domains that promote heterodimerization with the CH1 domain but minimize homodimerization with another Cκ domain. Exemplary modified human immunoglobulin Cκ domains are provided in SEQ ID NO: 142-178 of US Patent Application Publication No. 2013/0129723; SEQ ID NO: 160 (N29W V55A T70A), 161 (N29Y V55A T70A), 202 (T70E N29A N30A V55A), 167 (N30R V55A T70A), 168 (N30K V55A T70A), 170 (N30E V55A T70A), 172 ( V55R N29A N30A), 175 (N29W N30Y V55A T70E), 176 (N29Y N30Y V55A T70E), 177 (N30E V55A T70E), 178 (N30Y V55A T70E), 838 (N30D V55A T70E), 839 (N30M V55A T7 0E), 840 ( N30S V55A T70E) and 841 (N30F V55A T70E) of US Patent Application Publication No. 2013/0129723 (these sequences are incorporated herein by reference).

[00151] In certain embodiments, in addition to the mutations in the Cκ domains described herein, or as an alternative to these mutations, both immunoglobulin heterodimerization domains (i.e., immunoglobulin CH1 and CL domains) of the polypeptide heterodimer have mutations such that the resulting As a result, immunoglobulin heterodimerization domains form salt bridges (i.e., ionic interactions) between amino acid residues at the mutated sites. For example, the immunoglobulin heterodimerization domains of a polypeptide heterodimer may be a mutated CH1 domain in combination with a mutated Cκ domain. In the mutated CH1 domain, the valine at position 68 (V68) of the wild-type human CH1 domain is replaced by an amino acid residue having a negative charge (e.g., aspartate or glutamate), while the leucine at position 29 (L29) of the mutated human Cκ domain, in which the first one has been deleted arginine and the last cysteine are replaced by an amino acid residue that has a positive charge (for example, lysine, arginine or histidine). The charge interaction between the negatively charged amino acid residue of the resulting mutated CH1 domain and the positively charged amino acid residue of the resulting mutated Cκ domain forms a salt bridge that stabilizes the heterodimeric contact surface between the mutated CH1 and Cκ domains. Alternatively, wild-type V68 CH1 may be replaced by an amino acid residue having a positive charge, whereas L29 of the mutated human Cκ domain, in which the first arginine and last cysteine have been deleted, may be replaced by an amino acid residue having a negative charge. Exemplary mutated CH1 sequences in which V68 is replaced with a negative or positive amino acid are provided in SEQ ID NOs: 844 and 845 from US Patent Application Publication No. 2013/0129723 (these sequences are incorporated herein by reference). Exemplary mutated Cκ sequences in which L29 is replaced with a negatively or positively charged amino acid are provided in SEQ ID NOs: 842 and 843 from US Patent Application Publication No. 2013/0129723 (these sequences are incorporated herein by reference).

[00152] Positions of the human CH1 domain and L29 of the human Cκ domain other than V68 can be replaced with amino acids having opposite charges to create ionic interactions between amino acids in addition to or as an alternative to mutations in V68 of the CH1 domain and L29 of the Cκ domain. Such positions can be identified by any suitable method, including random mutagenesis, analysis of the crystal structure of the CH1-Cκ pair to identify amino acid residues at the CH1-Cκ interface, and identification of suitable positions among the amino acid residues at the CH1-Cκ interface using a set of criteria ( e.g., propensity to engage in ionic interactions, proximity to potential partner residue, etc.).

[00153] In certain embodiments, the polypeptide heterodimers of the present invention contain only one pair of immunoglobulin heterodimerization domains. For example, the first chain of a polypeptide heterodimer may contain a CH1 domain as an immunoglobulin heterodimerization domain, while the second chain may contain a CL domain (eg, Cκ or Cλ) as an immunoglobulin heterodimerization domain. Alternatively, the first strand may contain a CL domain (eg, Cκ or Cλ) as an immunoglobulin heterodimerization domain, while the second strand may contain a CH1 domain as an immunoglobulin heterodimerization domain. As described herein, the first and second chain immunoglobulin heterodimerization domains are capable of binding to form the heterodimeric protein of the present invention.

[00154] In some other embodiments, the heterodimeric proteins of the present invention may have two pairs of immunoglobulin heterodimerization domains. For example, the first strand of a heterodimer may contain two CH1 domains, while the second strand may have two CL domains binding to two CH1 domains in the first strand. Alternatively, the first strand may contain two CL domains, while the second strand may have two CH1 domains binding to two CL domains in the first strand. In certain embodiments, the first polypeptide chain comprises a CH1 domain and a CL domain, while the second polypeptide chain comprises a CL domain and a CH1 domain that bind to the CH1 domain and CL domain, respectively, of the first polypeptide chain.

[00155] In embodiments in which the heterodimeric protein contains only one heterodimerization pair (i.e., one immunoglobulin heterodimerization domain in each chain), the immunoglobulin heterodimerization domain of each chain may be amino-terminal to the immunoglobulin constant region of that chain. Alternatively, the immunoglobulin heterodimerization domain in each chain may be carboxy-terminal to the immunoglobulin constant region of that chain.

[00156] In embodiments in which the heterodimeric protein contains two heterodimerization pairs (i.e., two immunoglobulin heterodimerization domains on each chain), both immunoglobulin heterodimerization domains on each chain may be amino-terminal to the immunoglobulin constant region of that chain. Alternatively, both immunoglobulin heterodimerization domains in each chain may be carboxy-terminal to the immunoglobulin constant region of that chain. In additional embodiments, one immunoglobulin heterodimerization domain in each of the chains may be amino-terminal to the immunoglobulin constant region of that chain, while the other immunoglobulin heterodimerization domain of each of the chains may be carboxy-terminal to the immunoglobulin constant region of that chain. In other words, in these embodiments, the immunoglobulin constant region is located between two immunoglobulin heterodimerization domains of each chain.

[00157] As discussed herein, in some embodiments, the polypeptides and proteins (e.g., 5T4 binding polypeptides, 4-1BB binding polypeptides, and/or polyspecific binding proteins containing them) of the present invention further comprise an immunoglobulin constant region (also referred to as herein, constant region, Fc domain, Fc region, etc.) in a polypeptide chain. In some embodiments, the immunoglobulin constant region comprises the amino acid sequence of SEQ ID NO: 158, 160, or a variant thereof. Inclusion of an immunoglobulin constant region slows the clearance of the polypeptides and proteins of the present invention from the bloodstream after administration to a subject. Through mutations or other changes, the immunoglobulin constant region further allows for relatively easy modulation of the polypeptide's effector functions (eg, ADCC, ADCP, CDC, complement fixation, and Fc receptor binding), which may be increased or decreased depending on the disease being treated, as is known in the art and described herein. In certain embodiments, the polypeptides and proteins described herein contain an immunoglobulin constant region capable of mediating one or more of these effector functions. In other embodiments, one or more of these effector functions is reduced or absent in the immunoglobulin constant region of a polypeptide or protein described herein compared to the corresponding wild-type immunoglobulin constant region. For example, for dimeric 5T4-binding or 4-1BB-binding polypeptides designed to enhance effector cell activation, for example, by including a 4-1BB binding domain, the immunoglobulin constant region preferably has reduced effector function relative to the corresponding wild-type immunoglobulin constant region, or does not have such an effector function.

[00158] The immunoglobulin constant region present in the polypeptides and proteins of the present invention (e.g., 5T4-binding polypeptides, 4-1BB-binding polypeptides and/or polyspecific binding proteins containing them) may contain or be derived from part or all of: CH2 domain, CH3 domain, CH4 domain, or any combination thereof. For example, an immunoglobulin constant region may contain a CH2 domain, a CH3 domain, both CH2 and CH3 domains, both CH3 and CH4 domains, two CH3 domains, a CH4 domain, two CH4 domains, and a CH2 domain and part of a CH3 domain. In certain embodiments, the polypeptides or proteins described herein do not contain a CH1 domain.

[00159] A polypeptide or protein described herein may comprise a wild-type immunoglobulin CH2 domain or an altered immunoglobulin CH2 domain from certain classes or subclasses of immunoglobulins (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, or IgD) and from various species (including human, mouse, rat and other mammals). In certain embodiments, the CH2 domain of a polypeptide or protein described herein is a wild-type human immunoglobulin CH2 domain, such as the wild-type human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, or IgD CH2 domains as set forth in SEQ ID NO: 115, 199-201 and 195-197, respectively, from US Patent Application Publication No. 2013/0129723 (these sequences are incorporated herein by reference). In certain embodiments, the CH2 domain is the CH2 domain of wild-type human IgG1 as set forth in SEQ ID NO: 115 of U.S. Patent Application Publication No. US2013/0129723 (which sequence is incorporated herein by reference).

[00160] In certain embodiments, the altered CH2 region in a polypeptide or protein of the present invention contains or is at least 90%, at least 91%, at least 92%, or at least 93% sequence , at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the CH2 region of wild-type immunoglobulin, such as the CH2 region of wild-type human IgG1, IgG2, or IgG4, or mouse IgG2a (eg, IGHG2c).

[00161] The altered CH2 region of an immunoglobulin in a polypeptide or protein of the present invention (e.g., 5T4-binding polypeptides, 4-1BB-binding polypeptides, and/or polyspecific binding proteins containing them) can be derived from the CH2 region of various immunoglobulin isotypes, such as IgG1 , IgG2, IgG3, IgG4, IgA1, IgA2 and IgD, from various species (including human, mouse, rat and other mammals). In certain embodiments, the altered CH2 region of an immunoglobulin in a fusion protein of the present invention may be derived from the CH2 region of human IgG1, IgG2, or IgG4 or mouse IgG2a (e.g., IGHG2c), the sequences of which are set forth in SEQ ID NOs: 115, 199, 201, and 320 from US Patent Application Publication No. 2013/0129723 (these sequences are incorporated herein by reference). In certain embodiments, the altered CH2 domain of a polypeptide or protein described herein (e.g., 5T4 binding polypeptides, 4-1BB binding polypeptides, and/or polyspecific binding proteins containing them) is an altered human IgG1 CH2 domain containing mutations, which are known in the art to enhance or decrease immunological activities (ie, effector functions) such as ADCC, ADCP, CDC, complement fixation, Fc receptor binding, or any combination thereof.

[00162] In certain embodiments, the CH2 domain of a polypeptide or protein described herein is an altered CH2 region of an immunoglobulin (eg, an altered CH2 domain of human IgG1) containing one or more amino acid deletions or substitutions. In some embodiments, the CH2 domain comprises an asparagine amino acid substitution at position 297 (eg, an asparagine to alanine substitution). This amino acid substitution reduces or eliminates glycosylation at this site and inhibits efficient Fc binding to FcγR and C1q. The sequence of the altered CH2 domain of human IgG1 with an Asn to Ala substitution at position 297 is provided in SEQ ID NO: 324 of US Patent Application Publication No. 2013/0129723 (the said sequence is incorporated herein by reference). In some embodiments, the altered CH2 domain contains at least one substitution or deletion at positions 234 to 238. For example, the CH2 region of an immunoglobulin may contain a substitution at position 234, 235, 236, 237, or 238; provisions 234 and 235; provisions 234 and 236; provisions 234 and 237; provisions 234 and 238; provisions 234-236; provisions 234, 235 and 237; provisions 234, 236 and 238; provisions 234, 235, 237 and 238; provisions 236-238; or any other combination of two, three, four or five amino acids at positions 234-238. In some embodiments, the altered CH2 region contains one or more (e.g., two, three, four, or five) deletions of amino acids at positions 234-238, such as at one of position 236 or position 237, while a substitution is made at the other position . In certain embodiments, the amino acid residues at one or more positions 234-238 have been replaced by one or more alanine residues. In additional embodiments, only one of the amino acid residues at positions 234-238 has been deleted, while one or more remaining amino acids at positions 234-238 may be replaced with another amino acid (eg, alanine or serine).

[00163] In some embodiments, the above mutation(s) reduce or eliminate ADCC activity or the Fc receptor binding ability of a polypeptide containing an altered CH2 domain.

[00164] In some other embodiments, the CH2 domain of a polypeptide or protein described herein is an altered immunoglobulin CH2 region (e.g., an altered human IgG1 CH2 domain) containing one or more amino acid substitutions at positions 253, 310, 318, 320 , 322 and 331. For example, the CH2 region of an immunoglobulin may contain a substitution at positions 253, 310, 318, 320, 322 or 331, positions 318 and 320, positions 318 and 322, positions 318, 320 and 322, or any other combination of the two , three, four, five, or six amino acids at positions 253, 310, 318, 320, 322, and 331. In such embodiments, the above mutation(s) reduce or eliminate the CDC activity of the altered CH2 domain-containing polypeptide.

[00165] In some other embodiments, in addition to the amino acid substitution at position 297, the altered CH2 region of a polypeptide or protein described herein (e.g., an altered human IgG1 CH2 domain) may further comprise one or more (e.g., two, three, four or five) additional substitutions in provisions 234-238. For example, the CH2 region of an immunoglobulin may contain a substitution at positions 234 and 297, positions 234, 235 and 297, positions 234, 236 and 297, positions 234-236 and 297, positions 234, 235, 237 and 297, positions 234, 236, 238 and 297, positions 234, 235, 237, 238 and 297, positions 236-238 and 297, or any combination of two, three, four or five amino acids at positions 234-238 in addition to position 297. In addition or alternatively , the altered CH2 region may contain one or more (e.g., two, three, four, or five) deletions of amino acids at positions 234-238, such as position 236 or position 237. Additional mutation(s) reduce or eliminate ADCC activity or binding ability Fc receptor polypeptide containing an altered CH2 domain. In certain embodiments, the amino acid residues at one or more positions 234-238 have been replaced by one or more alanine residues. In additional embodiments, only one of the amino acid residues at positions 234-238 has been deleted, while one or more remaining amino acids at positions 234-238 may be replaced with another amino acid (eg, alanine or serine).

[00166] In certain embodiments, in addition to one or more (e.g., 2, 3, 4, or 5) amino acid substitutions at positions 234-238, the mutated CH2 region of a polypeptide or protein described herein (e.g., an altered CH2 domain human IgG1) in the fusion protein of the present invention may contain one or more (eg, 2, 3, 4, 5 or 6) additional amino acid substitutions (eg, alanine substitutions) at one or more positions involved in complement fixation (eg, positions I253, H310, E318, K320, K322 or P331). Examples of mutated immunoglobulin CH2 regions include the CH2 regions of human IgG1, IgG2, IgG4, and mouse IgG2a with alanine substitutions at positions 234, 235, 237 (if present), 318, 320, and 322. An example of a mutated immunoglobulin CH2 region is the CH2 region of mouse IGHG2c with alanine substitutions in L234, L235, G237, E318, K320 and K322.

[00167] In certain other embodiments, in addition to the amino acid substitution at position 297 and additional deletion(s) or substitution(s) at positions 234-238, an altered CH2 region of a polypeptide or protein described herein (e.g., an altered human CH2 domain IgG1) may further contain one or more (e.g., two, three, four, five, or six) additional substitutions at positions 253, 310, 318, 320, 322, and 331. For example, the CH2 region of an immunoglobulin may contain (1) a substitution at position 297, (2) one or more substitutions or deletions, or a combination thereof, at positions 234-238, and one or more (e.g., 2, 3, 4, 5, or 6) amino acid substitutions at positions I253, H310, E318, K320 , K322 and P331, for example one, two, three changes in positions E318, K320 and K322. The amino acids at the above positions may be replaced by alanine or serine.

[00168] In certain embodiments, the CH2 region of an immunoglobulin polypeptide or protein described herein contains: (i) an asparagine amino acid substitution at position 297 and one amino acid substitution at position 234, 235, 236, or 237; (ii) an amino acid substitution of asparagine at position 297 and amino acid substitutions at two positions from 234-237; (iii) an amino acid substitution of asparagine at position 297 and amino acid substitutions at three of positions 234-237; (iv) an amino acid substitution of asparagine at position 297, amino acid substitutions at positions 234, 235 and 237, and a deletion of amino acid at position 236; (v) amino acid substitutions at three positions from 234-237 and amino acid substitutions at positions 318, 320 and 322; or (vi) amino acid substitutions at three positions from 234-237, a deletion of an amino acid at position 236, and amino acid substitutions at positions 318, 320 and 322.

[00196] Exemplary altered immunoglobulin CH2 regions with asparagine amino acid substitutions at position 297 include: human IgG1 CH2 region with alanine substitutions at L234, L235, G237 and N297 and a deletion at G236 (SEQ ID NO: 325 from Patent Application Publication No. 2013/ 0129723, which sequence is incorporated herein by reference), the CH2 region of human IgG2 with alanine substitutions at V234, G236, and N297 (SEQ ID NO: 326 from US Patent Application Publication No. 2013/0129723, which sequence is incorporated herein by reference ), human IgG4 CH2 region with alanine substitutions at F234, L235, G237 and N297 and deletion at G236 (SEQ ID NO: 322 from US Patent Application Publication No. 2013/0129723, which sequence is incorporated herein by reference), CH2 region human IgG4 with alanine substitutions at F234 and N297 (SEQ ID NO: 343 from US Patent Application Publication No. US 2013/0129723, which sequence is incorporated herein by reference), the CH2 region of human IgG4 with alanine substitutions at L235 and N297 ( SEQ ID NO: 344 from US Patent Application Publication No. 2013/0129723, which sequence is incorporated herein by reference), the CH2 region of human IgG4 with alanine substitutions at G236 and N297 (SEQ ID NO: 345 from US Patent Application Publication No. 2013 /0129723, which sequence is incorporated herein by reference) and the CH2 region of human IgG4 with alanine substitutions at G237 and N297 (SEQ ID No. 346 of US Patent Application Publication No. 2013/0129723, which sequence is incorporated herein by reference). These CH2 regions can be used in a polypeptide of the present invention (eg, a 5T4 binding polypeptide and/or a 4-1BB bispecific polypeptide).

[00170] In certain embodiments, in addition to the amino acid substitutions described above, the altered CH2 region of a polypeptide or protein described herein (e.g., the altered CH2 domain of human IgG1) may contain one or more additional amino acid substitutions at one or more positions , different from the above provisions. Such amino acid substitutions may be conservative or non-conservative amino acid substitutions. For example, in certain embodiments, P233 may be replaced by E233 in the modified CH2 region of IgG2 (see, for example, SEQ ID NO: 326 of US Patent Application Publication No. 2013/0129723, which sequence is incorporated herein by reference). Additionally or alternatively, in certain embodiments, the altered CH2 region may contain one or more amino acid insertions, deletions, or both. The insertion(s), deletion(s), or substitution(s) may occur at any position of the CH2 region of an immunoglobulin, for example, at the N- or C-terminus of the CH2 region of a wild-type immunoglobulin resulting from binding of the CH2 region to another region (e.g. binding domain or immunoglobulin heterodimerization domain) via a hinge.

[00171] In certain embodiments, the altered CH2 domain of a polypeptide or protein described herein is a human IgG1 CH2 domain with alanine substitutions at positions 235, 318, 320, and 322 (i.e., a human IgG1 CH2 domain with L235A substitutions, E318A, K320A and K322A) (SEQ ID NO: 595 from US Patent Application Publication No. 2013/0129723, which sequence is incorporated herein by reference) and optionally the N297 mutation (eg, to alanine). In some other embodiments, the altered CH2 domain is a human IgG1 CH2 domain with alanine substitutions at positions 234, 235, 237, 318, 320, and 322 (i.e., a human IgG1 CH2 domain with substitutions L234A, L235A, G237A, E318A, K320A and K322A) (SEQ ID NO: 596 from US Patent Application Publication No. 2013/0129723, which sequence is incorporated herein by reference) and optionally the N297 mutation (eg, to alanine).

[00172] In some embodiments, the immunoglobulin constant region of a polypeptide or protein described herein (e.g., 5T4-binding polypeptides, 4-1BB-binding polypeptides, and/or polyspecific binding proteins containing them) comprises a human IgG1 CH2 domain containing substitutions L234A, L235A, G237A and K322A, according to the EU numbering system.

[00173] The CH3 domain, which may form the immunoglobulin constant region of a polypeptide or protein described herein (e.g., 5T4-binding polypeptides, 4-1BB-binding polypeptides, and/or polyspecific binding proteins containing them), may be the CH3 domain of an immunoglobulin wild-type or altered CH3 domain immunoglobulin from certain classes or subclasses of immunoglobulins (eg, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, IgM) from various species (including human, mouse, rat and other mammals). In some embodiments, the CH3 domain of a polypeptide described herein (e.g., 5T4-binding polypeptides, 4-1BB-binding polypeptides, and/or polyspecific binding proteins containing them) is the CH3 domain of wild-type human immunoglobulin, such as wild-type CH3 domains type human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE or IgM, as set forth in SEQ ID NO: 116, 208-210, 204-207 and 212, respectively, from US Patent Application Publication No. 2013/0129723 (these sequences are incorporated herein by reference). In certain embodiments, the CH3 domain is the CH3 domain of wild-type human IgG1 as set forth in SEQ ID NO: 116 of US Patent Application Publication No. 2013/0129723 (which sequence is incorporated herein by reference).

[00174] In certain embodiments, the CH3 domain of a polypeptide described herein (e.g., 5T4 binding polypeptides, 4-1BB binding polypeptides, and/or polyspecific binding proteins containing them) is an altered human immunoglobulin CH3 domain, such as an altered a CH3 domain based on or derived from the wild-type CH3 domain of human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, or IgM antibodies. For example, the altered CH3 domain may be the human IgG1 CH3 domain with one or two mutations at positions H433 and N434 (positions numbered according to the EU system). Mutations at such positions may be involved in complement fixation. In some other embodiments, the altered CH3 domain of a polypeptide described herein (e.g., 5T4 binding polypeptides, 4-1BB binding polypeptides, and/or polyspecific binding proteins containing them) may be the CH3 domain of human IgG1, but with one or more two amino acid substitutions at position F405 or Y407. Amino acids at such positions are involved in interaction with another CH3 domain. In certain embodiments, the altered CH3 domain of a polypeptide described herein may be an altered CH3 domain of human IgG1 in which the last lysine has been removed. The sequence of this altered CH3 domain is shown in SEQ ID NO: 761 from US Patent Application Publication No. 2013/0129723 (the said sequence is incorporated herein by reference).

[00175] In certain embodiments, a polypeptide or protein described herein (e.g., 5T4-binding polypeptides, 4-1BB-binding polypeptides, and/or polyspecific binding proteins containing them) contains a CH3 domain containing so-called "type" mutations. projections into depressions" (see Marvin and Zhu, Acta Pharmacologica Sinica 26:649-58, 2005; Ridgway et al. , Protein Engineering 9:617-21, 1966). More specifically, mutations can be introduced into each of the CH3 domains of each polypeptide chain such that the steric complementarity required for CH3/CH3 binding forces the two CH3 domains to pair with each other. For example, the CH3 domain in one single-chain polypeptide of a polypeptide heterodimer may contain the T366W mutation (a knob mutation that replaces a small amino acid with a larger one), and the CH3 domain in another single-chain polypeptide of a polypeptide heterodimer may contain the Y407A mutation (a trench mutation that replaces a large one). amino acid to a smaller one). Other exemplary peak-to-trench mutations include (1) the T366Y mutation in one CH3 domain and Y407T in another CH3 domain, and (2) the T366W mutation in one CH3 domain and the T366S, L368A, and Y407V mutations in another CH3 domain.

[00176] The CH4 domain, which may form the immunoglobulin constant region of a polypeptide or protein described herein (e.g., 5T4-binding polypeptides, 4-1BB-binding polypeptides, and/or polyspecific binding proteins containing them), may be an immunoglobulin CH4 domain wild type or modified immunoglobulin CH4 domain from IgE or IgM molecules. In certain embodiments, the CH4 domain of a polypeptide described herein is the CH4 domain of a wild-type human immunoglobulin, such as the wild-type CH4 domains of human IgE and IgM molecules as set forth in SEQ ID NOs: 213 and 214, respectively, from Patent Publication US Application No. 2013/0129723 (these sequences are incorporated herein by reference). In certain embodiments, the CH4 domain of a polypeptide described herein is an altered CH4 domain of a human immunoglobulin, such as an altered CH4 domain based on or derived from the CH4 domain of human IgE or IgM molecules having mutations that increase or decrease the immunological activity associated , as is known, with the Fc region of IgE or IgM.

[00177] In certain embodiments, the immunoglobulin constant region of a polypeptide or protein described herein (e.g., 5T4-binding polypeptides, 4-1BB-binding polypeptides, and/or polyspecific binding proteins containing them) contains a combination of CH2, CH3, or CH4 domains (ie, more than one constant region domain is selected from CH2, CH3 and CH4). For example, an immunoglobulin constant region may contain CH2 and CH3 domains or CH3 and CH4 domains. In some other embodiments, the immunoglobulin constant region may contain two CH3 domains and no CH2 or CH4 domains (ie, only two or more CH3). The multiple constant region domains constituting the immunoglobulin constant region of the polypeptides described herein may be based on or derived from the same immunoglobulin molecule, or from immunoglobulin molecules of the same class or subclass. In certain embodiments, the immunoglobulin constant region is CH2-CH3 IgG (eg, CH2-CH3 IgG1, CH2-CH3 IgG2, and CH2-CH3 IgG4) and may be human CH2CH3 (eg, human IgG1, IgG2, and IgG4). For example, in certain embodiments, the immunoglobulin constant region of a polypeptide described herein comprises (1) the CH2 and CH3 domains of wild-type human IgG1, (2) the CH2 domains of human IgG1 with an N297A substitution (i.e., CH2(N297A)), and CH3 of wild-type human IgG1, or (3) CH2(N297A) of human IgG1 and altered CH3 of human IgG1 whose last lysine has been removed. Alternatively, multiple constant region domains of a polypeptide or protein described herein (e.g., 5T4-binding polypeptides, 4-1BB-binding polypeptides, and/or polyspecific binding proteins containing them) may be based on or derived from different immunoglobulin molecules, or from immunoglobulin molecules of different classes or subclasses. For example, in certain embodiments, the immunoglobulin constant region comprises both a CH3 domain of human IgM and a CH3 domain of human IgG1. The multiple constant region domains constituting the immunoglobulin constant region of a polypeptide described herein may be directly linked to each other or may be linked to each other by one or more (eg, about 2 to about 10) amino acids.

[00178] Exemplary immunoglobulin constant regions that may be used in a polypeptide or protein described herein (e.g., 5T4 binding polypeptides, 4-1BB binding polypeptides, and/or polyspecific binding proteins containing them) are provided in SEQ ID NO: 305-309, 321, 323, 341, 342 and 762 from US Patent Application Publication No. 2013/0129723 (these sequences are incorporated herein by reference). Additional exemplary immunoglobulin constant regions that may be used in a polypeptide or protein described herein are presented below in Table 4.

Table 4. Exemplary Immunoglobulin Constant Regions

Name Amino acid sequence SEQ ID NO: SS-Fc domain SSEPKSSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 158 Delta SS-Fc domain EPKSSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 160

[00179] In certain embodiments, the immunoglobulin constant regions of each of the polypeptide chains of a homodimeric or heterodimeric protein described herein (e.g., 5T4-binding polypeptides, 4-1BB-binding polypeptides, and/or polyspecific binding proteins containing them) are identical to each other. In some other embodiments, the immunoglobulin constant region of one polypeptide chain of the heterodimer protein is different from the immunoglobulin constant region of the other polypeptide chain of the heterodimer. For example, one immunoglobulin constant region of a heterodimeric protein may contain a CH3 domain with a knob mutation, while another immunoglobulin constant region of a heterodimeric protein may contain a CH3 domain with a trench mutation.

[00180] In some embodiments, the polypeptides of the present invention may comprise an immunoglobulin constant region containing any of the mutations described above, and a binding domain containing one or more amino acid mutations relative to the amino acid sequence of the original binding domain. For example, in some embodiments, the polypeptides of the present invention may comprise an immunoglobulin constant region comprising one or more mutations L234A, L235A, G237A and K322A in the CH2 domain of human IgG1, and a 5T4 binding domain comprising a VH domain selected from the group consisting of SEQ ID NOs: 38 and 46, and a VL domain selected from the group consisting of SEQ ID NOs: 44, 48, and 50. In some embodiments, the polypeptides of the present invention may contain an immunoglobulin constant region containing one or more mutations L234A, L235A , G237A and K322A in the CH2 domain of human IgG1, and a 41BB binding domain comprising the _VH domain amino acid sequence of SEQ ID NO: 14 and the _VL domain amino acid sequence of SEQ ID NO: 16. In some embodiments The polypeptides of the present invention may comprise an immunoglobulin constant region containing one or more of the L234A, L235A, G237A and K322A mutations in the CH2 domain of human IgG1, and a 5T4 binding domain comprising a _VH domain selected from the group consisting of SEQ ID NO: 38 and 46, and a V _L domain selected from the group consisting of SEQ ID NOs: 44, 48 and 50; and a 41BB-binding domain comprising the amino acid sequence of the V _H domain being SEQ ID NO: 14 and the amino acid sequence of the V _L domain being SEQ ID NO: 16.

[00181] The polypeptides and proteins described herein (e.g., 5T4-binding polypeptides, 4-1BB-binding polypeptides, and/or polyspecific binding proteins containing them) can be prepared using scaffolding, as generally disclosed in patent application publications US Nos. 2013/0129723 and 2013/0095097, each of which is incorporated herein by reference in its entirety. The polypeptides described herein may contain two non-identical polypeptide chains, each of which contains an immunoglobulin heterodimerization domain. Immunoglobulin heterodimerization domains at the contact surface differ. In one embodiment, the immunoglobulin heterodimerization domain comprises a CH1 domain or a derivative thereof. In yet another embodiment, the immunoglobulin heterodimerization domain comprises a CL domain or a derivative thereof. In one embodiment, the CL domain is a Cκ or Cλ isotype, or a derivative thereof.

[00182] In some embodiments, the polypeptides and proteins described herein (e.g., 5T4 binding polypeptides, 4-1BB binding polypeptides, and/or polyspecific binding proteins containing them) may have improved characteristics compared to other 5T4 binding polypeptides or 4-1BB-binding polypeptides. For example, the 5T4-binding polypeptides, 4-1BB-binding polypeptides, or polyspecific proteins containing them of the present invention may have a reduced isoelectric point compared to the isoelectric point of another 5T4-binding polypeptide and/or 4-1BB-binding polypeptide. The "isoelectric point" or "pI" is the pH at which the net charge is zero. The isoelectric point of a protein can be measured by any suitable method, for example, using analytical capillary chromatography using the isoelectric focusing method.

[00183] In some embodiments, the polypeptides and proteins described herein (e.g., 5T4-binding polypeptides, 4-1BB-binding polypeptides, and/or polyspecific binding proteins containing them) may bind 5T4 (e.g., human 5T4) and/or or 4-1BB with higher affinity compared to known 5T4 and/or 4-1BB binding domains and/or the original 5T4 and/or 4-1BB binding domain or protein. In some embodiments, the dissociation constant of the 5T4 and/or 4-1BB binding domain or polypeptide may be approximately 2-5 nM. In certain embodiments, the dissociation rate of the 5T4 and/or 4-1BB binding domain or polypeptide can be reduced by 4-10 times compared to the dissociation rate of a known antibody or scFv construct, or the original 5T4 and/or 4-1BB binding domain or protein.

[00184] In some embodiments, the polypeptides and proteins described herein (e.g., 5T4-binding polypeptides, 4-1BB-binding polypeptides, and/or polyspecific binding proteins containing them) may have low levels of high molecular weight aggregates formed during recombinant expression polypeptide or protein. In some embodiments, the polypeptides and proteins described herein (e.g., 5T4-binding polypeptides, 4-1BB-binding polypeptides, and/or polyspecific binding proteins containing them) may have longer-term stability in human serum, depending on the combination of domains, present in a polypeptide or protein.

5T4-binding domains and proteins containing them

[00185] In some embodiments, the present invention provides 5T4 binding domains that specifically bind to 5T4 (eg, human 5T4). In some embodiments, the present invention further provides polypeptides containing a 5T4 binding domain (eg, 5T4 binding polypeptides). In some variations, the 5T4-binding polypeptide contains a hinge region, which is carboxy-terminal to the 5T4-binding domain, and an immunoglobulin constant region. In further variations, the 5T4 binding polypeptide comprises a carboxy-terminal binding domain, a linker carboxy-terminal to the immunoglobulin constant region, and a second binding domain carboxy-terminal to the carboxy-terminal linker. In other variations, the 5T4-binding polypeptide contains a hinge region amino-terminal to the polypeptide containing the 5T4-binding domain and an immunoglobulin constant region amino-terminal to the hinge region.

[00186] In some embodiments, the 5T4 binding domains described herein bind an epitope located on the extracellular domain of 5T4 (eg, the epitope contained in SEQ ID NO: 168). In certain aspects, the epitope is discontinuous and/or conformational.

[00187] A 5T4 binding domain polypeptide may specifically bind to human 5T4 and comprise a heavy chain CDR1 (HCDR1), HCDR2, HCDR3, a light chain CDR1 (LCDR1), LCDR2, and LCDR3, wherein HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 30, 52 and 60; HCDR2 contains an amino acid sequence selected from the group consisting of SEQ ID NO: 32 and 62; HCDR3 contains the amino acid sequence SEQ ID NO: 34; LCDR1 contains an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 42 and 54; LCDR2 contains the amino acid sequence SEQ ID NO: 10; and LCDR3 contains the amino acid sequence of SEQ ID NO: 36.

[00188] In particular embodiments, the 5T4 binding domain polypeptide may specifically bind to human 5T4 and comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein (a) HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 30; (b) HCDR2 contains the amino acid sequence shown in SEQ ID NO: 32; (c) HCDR3 contains the amino acid sequence shown in SEQ ID NO: 34; (d) LCDR1 contains the amino acid sequence shown in SEQ ID NO: 8; LCDR2 contains the amino acid sequence shown in SEQ ID NO: 10; and (f) LCDR3 contains the amino acid sequence set forth in SEQ ID NO: 36. In other particular embodiments, the 5T4 binding domain polypeptide specifically binds to human 5T4 and comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein (a) HCDR1 contains the amino acid sequence set forth in SEQ ID NO: 30; (b) HCDR2 contains the amino acid sequence shown in SEQ ID NO: 32; (c) HCDR3 contains the amino acid sequence shown in SEQ ID NO: 34; (d) LCDR1 contains the amino acid sequence shown in SEQ ID NO: 42; (e) LCDR2 contains the amino acid sequence shown in SEQ ID NO: 10; and (f) LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 36. In other particular embodiments, the 5T4 binding domain polypeptide specifically binds to human 5T4 and comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein (a) HCDR1 contains the amino acid sequence shown in SEQ ID NO: 52; (b) HCDR2 contains the amino acid sequence shown in SEQ ID NO: 32; (c) HCDR3 contains the amino acid sequence shown in SEQ ID NO: 34; (d) LCDR1 contains the amino acid sequence shown in SEQ ID NO: 54; (e) LCDR2 contains the amino acid sequence shown in SEQ ID NO: 10; and (f) LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 36. In other particular embodiments, the 5T4 binding domain polypeptide specifically binds to human 5T4 and comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein (a) HCDR1 contains the amino acid sequence shown in SEQ ID NO: 60; (b) HCDR2 contains the amino acid sequence shown in SEQ ID NO: 62; (c) HCDR3 contains the amino acid sequence shown in SEQ ID NO: 34; (d) LCDR1 contains the amino acid sequence shown in SEQ ID NO: 54; (e) LCDR2 contains the amino acid sequence shown in SEQ ID NO: 10; and (f) LCDR3 contains the amino acid sequence set forth in SEQ ID NO: 36.

[00189] In certain embodiments, the 5T4 binding domain polypeptide contains or is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least at least about 99%, at least about 99.5%, or 100% identical to the amino acid sequence of a light chain variable region (V _L ) selected from the group consisting of SEQ ID NO: 40, 44, 48, 50, 58 , 68 and 70, where the specified polypeptide is a polyspecific polypeptide in the scFv-Fc-scFv format and contains a Y to F substitution in LCDR1 at position 99 V _L and/or an F to S substitution in FR3 at position 148 V _L . In certain embodiments, the 5T4 binding domain-containing polypeptide comprises a light chain variable region (V _L ) amino acid sequence selected from the group consisting of SEQ ID NOs: 40, 44, 48, 50, 58, 68, and 70. In certain embodiments In embodiments, the 5T4 binding domain polypeptide comprises a heavy chain variable region ( _VH ) amino acid sequence selected from the group consisting of SEQ ID NOs: 38, 46, 56, and 64.

[00190] In certain embodiments, the 5T4 binding domain polypeptide comprises a V _H region comprising the amino acid sequence of SEQ ID NO: 38 and a V _L region containing the amino acid sequence of SEQ ID NO: 40. In certain embodiments, the 5T4 binding domain polypeptide comprises a V _H region containing the amino acid sequence of SEQ ID NO: 38, and a V _L region containing the amino acid sequence of SEQ ID NO: 44. In certain embodiments, the 5T4 binding domain polypeptide comprises a V _H region containing the amino acid sequence of SEQ ID NO: 46, and a V _L region containing the amino acid sequence of SEQ ID NO: 48. In certain embodiments, the 5T4 binding domain polypeptide comprises a V _H region containing the amino acid sequence of SEQ ID NO: 46 and a V _L region containing the amino acid sequence of SEQ ID NO: 50 In certain embodiments, the 5T4 binding domain polypeptide comprises a V _H region comprising the amino acid sequence of SEQ ID NO: 56 and a V _L region containing the amino acid sequence of SEQ ID NO: 58. In certain embodiments, the 5T4 binding domain polypeptide comprises a V region _H containing the amino acid sequence of SEQ ID NO: 64, and a V _L region containing the amino acid sequence of SEQ ID NO: 58. In certain embodiments, the 5T4 binding domain polypeptide comprises a V _H region containing the amino acid sequence of SEQ ID NO: 38, and a region V _L containing the amino acid sequence of SEQ ID NO: 68. In certain embodiments, the 5T4 binding domain polypeptide comprises a V _H region containing the amino acid sequence of SEQ ID NO: 46 and a V _L region containing the amino acid sequence of SEQ ID NO: 70.

[00191] Exemplary 5T4 binding domain sequences are presented in Table 5.

Table 5. Amino acid and DNA sequences of the CDR and variable region of the 5T4-binding domain polypeptide

Design Component DNA sequence SEQ ID NO: DNA sequences Amino acid sequence SEQ ID NO: amino acid sequence ALG.APV-178
ALG.APV-208 HCDR1 ggcttcacattcagcagctatgct 29 GFTFSSYA thirty HCDR2 atctccggcagcggcggaagcacc 31 ISGSGGST 32 HCDR3 gccaggtactatggcggctactactccgcctggatggactac 33 ARYYGGYYSAWMDY 34 LCDR1 cagtccatctccagctat 7 QSISSY 8 LCDR2 gccgcttcc 9 A.A.S. 10 LCDR3 cagcagacctatggctacctgcacacc 35 QQTYGYLHT 36 V _H gaagtgcagctgctggagtccggaggaggactggtgcagcctggcggaagcctgaggctgagctgcgctgcctccggcttcacattcagcagctatgctatgagctgggtgaggcaagcccctggaaagggcctggagtgggtgtccgctatctccggcagcggcggaagcacctactacgctgactccgtcaagggcagg ttcaccatcagccgggacaacagcaagaacaccctgtacctgcagatgaatagcctcagggctgaagacaccgctgtgtactactgcgccaggtactatggcggctactactccgcctggatggactactggggacagggcacactggtgaccgtgtccagc 37 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSS 38 V _L gatattcagatgacacagtcccctagctccctgtccgccagcgtgggagatcgggtgaccatcacctgcagggccagccagtccatctccagctatttaaactggtaccagcagaagcctggaaaggctcccaagctgctgatctacgccgcttccagcctccagagcggcgtgcctagcaggttctccggctccggaagcggaacagact tcaccctgaccatcagctccctgcagcccgaggactccgctacctactactgccagcagacctatggctacctgcacaccttcggccagggcacaaagctggagatcaag 39 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDSATYYCQQTYGYLHTFGQGTKLEIK 40 ALG.APV-179
ALG.APV-209
ALG.APV-222 HCDR1 ggcttcacattcagcagctatgct 29 GFTFSSYA thirty HCDR2 atctccggcagcggcggaagcacc 31 ISGSGGST 32 HCDR3 gccaggtactatggcggctactactccgcctggatggactac 33 ARYYGGYYSAWMDY 34 LCDR1 cagtccatctccagcttc 41 QSISSF 42 LCDR2 gccgcttcc 9 A.A.S. 10 LCDR3 cagcagacctatggctacctgcacacc 35 QQTYGYLHT 36 V _H gaagtgcagctgctggagtccggaggaggactggtgcagcctggcggaagcctgaggctgagctgcgctgcctccggcttcacattcagcagctatgctatgagctgggtgaggcaagcccctggaaagggcctggagtgggtgtccgctatctccggcagcggcggaagcacctactacgctgactccgtcaagggcagg ttcaccatcagccgggacaacagcaagaacaccctgtacctgcagatgaatagcctcagggctgaagacaccgctgtgtactactgcgccaggtactatggcggctactactccgcctggatggactactggggacagggcacactggtgaccgtgtccagc 37 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSS 38 V _L gatattcagatgacacagtcccctagctccctgtccgccagcgtgggagatcgggtgaccatcacctgcagggccagccagtccatctccagcttcttaaactggtaccagcagaagcctggaaaggctcccaagctgctgatctacgccgcttccagcctccagagcggcgtgcctagcaggttctccggctccggaagcggaacag acttcaccctgaccatcagctccctgcagcccgaggactccgctacctactactgccagcagacctatggctacctgcacaccttcggccagggcacaaagctggagatcaag 43 DIQMTQSPSSLSASVGDRVTITCRASQSISSFLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDSATYYCQQTYGYLHTFGQGTKLEIK 44 ALG.APV-187
ALG.APV-210
ALG.APV-223 HCDR1 ggcttcacattcagcagctatgct 29 GFTFSSYA thirty HCDR2 atctccggcagcggcggaagcacc 31 ISGSGGST 32 HCDR3 gccaggtactatggcggctactactccgcctggatggactac 33 ARYYGGYYSAWMDY 34 LCDR1 cagtccatctccagcttc 41 QSISSF 42 LCDR2 gccgcttcc 9 A.A.S. 10 LCDR3 cagcagacctatggctacctgcacacc 35 QQTYGYLHT 36 V _H gaagtgcagctgctggagtccggaggaggactggtgcagcctggcggaagcctgaggctgagctgcgctgcctccggcttcacattcagcagctatgctatgagctgggtgaggcaagcccctggaaagtgcctggagtgggtgtccgctatctccggcagcggcggaagcacctactacgctgactccgtcaagggca ggttcaccatcagccgggacaacagcaagaacaccctgtacctgcagatgaatagcctcagggctgaagacaccgctgtgtactactgcgccaggtactatggcggctactccgcctggatggactactggggacagggcacactggtgaccgtgtccagc 45 EVQLLESGGGLVQPGGSLRLSCAASGFFSSYAMSWVRQAPGKCLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSS 46 V _L gatattcagatgacacagtcccctagctccctgtccgccagcgtgggagatcgggtgaccatcacctgcagggccagccagtccatctccagcttcttaaactggtaccagcagaagcctggaaaggctcccaagctgctgatctacgccgcttccagcctccagagcggcgtgcctagcaggttctccggctccggaagcggaacag acttcaccctgaccatcagctccctgcagcccgaggactccgctacctactactgccagcagacctatggctacctgcacaccttcggctgcggcacaaagctggagatcaag 47 DIQMTQSPSSLSASVGDRVTITCRASQSISSFLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDSATYYCQQTYGYLHTFGCGTKLEIK 48 ALG.APV-191 HCDR1 ggcttcacattcagcagctatgct 29 GFTFSSYA thirty HCDR2 atctccggcagcggcggaagcacc 31 ISGSGGST 32 HCDR3 gccaggtactatggcggctactactccgcctggatggactac 33 ARYYGGYYSAWMDY 34 LCDR1 cagtccatctccagcttc 41 QSISSF 42 LCDR2 gccgcttcc 9 A.A.S. 10 LCDR3 cagcagacctatggctacctgcacacc 35 QQTYGYLHT 36 V _H gaagtgcagctgctggagtccggaggaggactggtgcagcctggcggaagcctgaggctgagctgcgctgcctccggcttcacattcagcagctatgctatgagctgggtgaggcaagcccctggaaagtgcctggagtgggtgtccgctatctccggcagcggcggaagcacctactacgctgactccgtcaagggca ggttcaccatcagccgggacaacagcaagaacaccctgtacctgcagatgaatagcctcagggctgaagacaccgctgtgtactactgcgccaggtactatggcggctactccgcctggatggactactggggacagggcacactggtgaccgtgtccagc 45 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKCLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSS 46 V _L gatattcagatgacacagtcccctagctccctgtccgccagcgtgggagatcgggtgaccatcacctgcagggccagccagtccatctccagcttcttaaactggtaccagcagaagcctggaaaggctcccaagctgctgatctacgccgcttccagcctccagagcggcgtgcctagcaggttctccggctccggaagcggaacag acttcaccctgaccatcagctccctgcagcccgaggacttcgctacctactactgccagcagacctatggctacctgcacaccttcggctgcggcacaaagctggagatcaag 49 DIQMTQSPSSLSASVGDRVTITCRASQSISSFLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYGYLHTFGCGTKLEIK 50 ALG.APV-196
ALG.APV-198 HCDR1 ggcttcgacttcgagagctatgct 51 GFDFESYA 52 HCDR2 atctccggcagcggcggaagcacc 31 ISGSGGST 32 HCDR3 gccaggtactatggcggctactactccgcctggatggactac 33 ARYYGGYYSAWMDY 34 LCDR1 cagtccatcaggagcgcc 53 QSIRSA 54 LCDR2 gccgcttcc 9 A.A.S. 10 LCDR3 cagcagacctatggctacctgcacacc 35 QQTYGYLHT 36 V _H gaagtgcagctgctggagtccggaggaggactggtgcagcctggcggaagcctgaggctgagctgcgctgcctccggcttcgacttcgagagctatgctatgagctgggtgaggcaagcccctggaaagtgcctggagtgggtgtccgctatctccggcagcggcggaagcacctactacgctgactccgtcaaggg caggttcaccatcagccgggacaacagcaagaacaccctgtacctgcagatgaatagcctcagggctgaagacaccgctgtgtactactgcgccaggtactatggcggctactccgcctggatggactactggggacagggcacactggtgaccgtgtccagc 55 EVQLLESGGGLVQPGGSLRLSCAASGFDFESYAMSWVRQAPGKCLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSS 56 V _L gatattcagatgacacagtcccctagctccctgtccgccagcgtgggagatcgggtgaccatcacctgcagggccagccagtccatcaggagcgccctgaactggtaccagcagaagcctggaaaggctcccaagctgctgatctacgccgcttccagcctccagagcggcgtgcctagcaggttctccggctccggaagcggaacag acttcaccctgaccatcagctccctgcagcccgaggacttcgctacctactactgccagcagacctatggctacctgcacaccttcggctgcggcacaaagctggagatcaag 57 DIQMTQSPSSLSASVGDRVTITCRASQSIRSALNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYGYLHTFGCGTKLEIK 58 ALG.APV-199 HCDR1 ggcttcgacttcgacagctatgct 59 GFFDDSYA 60 HCDR2 atctccggcaggggcggaagcacc 61 ISGRGGST 62 HCDR3 gccaggtactatggcggctactactccgcctggatggactac 33 ARYYGGYYSAWMDY 34 LCDR1 cagtccatcaggagcgcc 53 QSIRSA 54 LCDR2 gccgcttcc 9 A.A.S. 10 LCDR3 cagcagacctatggctacctgcacacc 35 QQTYGYLHT 36 V _H gaagtgcagctgctggagtccggaggaggactggtgcagcctggcggaagcctgaggctgagctgcgctgcctccggcttcgacttcgacagctatgctatgagctgggtgaggcaagcccctggaaagtgcctggagtgggtgtccgctatctccggcaggggcggaagcacctactacgctgactccgtcaagggca ggttcaccatcagccgggacaacagcaagaacaccctgtacctgcagatgaatagcctcagggctgaagacaccgctgtgtactactgcgccaggtactatggcggctactccgcctggatggactactggggacagggcacactggtgaccgtgtccagc 63 EVQLLESGGGLVQPGGSLRLSCAASGFDFDSYAMSWVRQAPGKCLEWVSAISGRGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSS 64 V _L gatattcagatgacacagtcccctagctccctgtccgccagcgtgggagatcgggtgaccatcacctgcagggccagccagtccatcaggagcgccctgaactggtaccagcagaagcctggaaaggctcccaagctgctgatctacgccgcttccagcctccagagcggcgtgcctagcaggttctccggctccggaagcggaacag acttcaccctgaccatcagctccctgcagcccgaggacttcgctacctactactgccagcagacctatggctacctgcacaccttcggctgcggcacaaagctggagatcaag 57 DIQMTQSPSSLSASVGDRVTITCRASQSIRSALNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYGYLHTFGCGTKLEIK 58 ALG.APV-004 HCDR1 ggattcacctttagcagctatgcc 29 GFTFSSYA thirty HCDR2 attagtggtagtggtggtagcaca 31 ISGSGGST 32 HCDR3 gcgcgctactacggtggttactactctgcttggatggactat 33 ARYYGGYYSAWMDY 34 LCDR1 cagagcattagcagctat 7 QSISSY 8 LCDR2 gctgcatcc 9 A.A.S. 10 LCDR3 caacagacttacggttacctgcacact 35 QQTYGYLHT 36 V _H gaggtgcagctgctcgagagcgggggaggcttggtacagcctggggggtccctgcgcctctcctgtgcagccagcggattcacctttagcagctatgccatgagctgggtccgccaggctccagggaagtgtctggagtgggtctcagctattagtggtagtggtggtagcacatactatgcagactccgtgaagggccggttcacca tctcccgtgacaattccaagaacacgctgtatctgcaaatgaacagcctgcgtgccgaggacacggctgtatattattgtgcgcgctactacggtggttactactctgcttggatggactattggggccagggaaccctggtcaccgtctcctca 45 EVQLLESGGGLVQPGGSLRLSCAASGFFSSYAMSWVRQAPGKCLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSS 46 V _L gacatccagatgacccagtctccatcctccctgagcgcatctgtaggagaccgcgtcaccatcacttgccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtttgcaaagtggggtcccatcacgtttcagtggcagtggaagcgggaca gatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttattactgtcaacagacttacggttacctgcacacttttggctgtgggaccaggctggagatcaaa 65 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYGYLHTFGCGTRLEIK 66 ALG.APV-006
ALG.APV-010 HCDR1 ggattcacctttagcagctatgcc 29 GFTFSSYA thirty HCDR2 attagtggtagtggtggtagcaca 31 ISGSGGST 32 HCDR3 gcgcgctactacggtggttactactctgcttggatggactat 33 ARYYGGYYSAWMDY 34 LCDR1 cagagcattagcagctat 7 QSISSY 8 LCDR2 gctgcatcc 9 A.A.S. 10 LCDR3 caacagacttacggttacctgcacact 35 QQTYGYLHT 36 V _H gaggtgcagctgttggagagcgggggaggcttggtacagcctggggggtccctgcgcctctcctgtgcagccagcggattcacctttagcagctatgccatgagctgggtccgccaggctccaggaaggggctggagtgggtctcagctattagtggtagtggtggtagcacatactatgcagactccgtgaagggccggttcaccat ctcccgtgacaattccaagaacacgctgtatctgcaaatgaacagcctgcgtgccgaggacacggctgtatattattgtgcgcgctactacggtggttactactctgcttggatggactattggggccagggaaccctggtcaccgtctcctca 37 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSS 38 V _L gacatccagatgacccagtctccatcctccctgagcgcatctgtaggagaccgcgtcaccatcacttgccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtttgcaaagtggggtcccatcacgtttcagtggcagtggaagcgggaca gatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttattactgtcaacagacttacggttacctgcacacttttggccaggggaccaagctggagatcaaa 67 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYGYLHTFGQGTKLEIK 68 ALG.APV-014
ALG.APV-018 HCDR1 ggattcacctttagcagctatgcc 29 GFTFSSYA thirty HCDR2 attagtggtagtggtggtagcaca 31 ISGSGGST 32 HCDR3 gcgcgctactacggtggttactactctgcttggatggactat 33 ARYYGGYYSAWMDY 34 LCDR1 cagagcattagcagctat 7 QSISSY 8 LCDR2 gctgcatcc 9 A.A.S. 10 LCDR3 caacagacttacggttacctgcacact 35 QQTYGYLHT 36 V _H gaggtgcagctgttggagagcgggggaggcttggtacagcctggggggtccctgcgcctctcctgtgcagccagcggattcacctttagcagctatgccatgagctgggtccgccaggctccagggaagtgcctggagtgggtctcagctattagtggtagtggtggtagcacatactatgcagactccgtgaagggccggttcacca tctcccgtgacaattccaagaacacgctgtatctgcaaatgaacagcctgcgtgccgaggacacggctgtatattattgtgcgcgctactacggtggttactactctgcttggatggactattggggccagggaaccctggtcaccgtctcctca 45 EVQLLESGGGLVQPGGSLRLSCAASGFFSSYAMSWVRQAPGKCLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSS 46 V _L gacatccagatgacccagtctccatcctccctgagcgcatctgtaggagaccgcgtcaccatcacttgccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtttgcaaagtggggtcccatcacgtttcagtggcagtggaagcgggaca gatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttattactgtcaacagacttacggttacctgcacacttttggctgcgggaccaagctggagatcaaa 69 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYGYLHTFGCGTKLEIK 70

[00192] In some embodiments, the present invention provides polyspecific polypeptides (eg, bispecific polypeptides) containing a 5T4 binding domain. In such embodiments, the 5T4 binding protein or polypeptide may comprise one or more additional binding domains (eg, a second binding domain) that bind a target other than 5T4. These other binding domains may contain, for example, a particular cytokine or molecule that targets the binding domain polypeptide, for example, to a specific cell type, toxin, additional cellular receptor or antibody. A polyspecific 5T4-binding polypeptide or protein may contain two binding domains (the domains may be designed to specifically bind to the same or different targets), a hinge region, a linker (eg, a carboxy-terminal or amino-terminal linker), and an immunoglobulin constant region. The polyspecific 5T4 binding protein may be a homodimeric protein containing two identical polypeptides linked by a disulfide bond. In some embodiments, the 5T4-binding domains may be derived from a 5T4-binding monoclonal antibody.

[00193] In some embodiments, the 5T4-binding polypeptide is capable of forming a heterodimer with a second polypeptide chain and contains a hinge region (a) immediately amino-terminal to an immunoglobulin constant region (e.g., amino-terminal to a CH2 domain if the immunoglobulin constant region includes CH2 domains and CH3, or amino-terminal to the CH3 domain if the immunoglobulin subregions include CH3 and CH4 domains), (b) located between and connecting the binding domain (e.g., scFv) and the immunoglobulin heterodimerization domain, (c) located between the immunoglobulin heterodimerization domain and the constant region of an immunoglobulin (for example, when the immunoglobulin constant region includes the CH2 and CH3 domains or the CH3 and CH4 domains) and connecting them, (d) located between and connecting the immunoglobulin constant region and the binding domain, (e) at the amino terminus of the polypeptide chain or ( f) at the carboxy end of the polypeptide chain. A polypeptide chain containing a hinge region as described herein will be capable of binding to another polypeptide chain to form a heterodimeric protein as provided herein, and the resulting heterodimer will contain a binding domain retaining its target specificity or its specific target binding affinity .

[00194] In some embodiments, the 5T4 binding polypeptide provided herein is a polypeptide comprising two scFvs. In some embodiments, the two scFvs contain 5T4 binding domains. In certain embodiments, the two scFvs contain identical 5T4 binding domains. In other embodiments, the two scFvs contain different 5T4 binding domains. In other embodiments, the polypeptide comprises a 5T4 binding domain as a first scFv and an effector cell binding domain as a second scFv. For example, the effector cell binding domain may be a 4-1BB specific scFv.

[00195] In some embodiments, the 5T4 binding polypeptide provided herein comprises at least about 82% anti-5T4 scFv, at least about 85%, at least about 87%, at least about by 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96 %, at least about 97%, at least about 98%, at least about 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 118, 120, 122, 124 , 126, 128, 130, 132, 134 and 170, where the specified polypeptide is a polyspecific polypeptide in the scFv-Fc -scFv format and contains a Y to F substitution in LCDR1 at position 99 V _L to 5T4 and/or an F to S substitution in FR3 in position 148 V _L to 5T4. In some embodiments, a 5T4 binding polypeptide provided herein comprises an anti-5T4 scFv comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 118, 120, 122, 124, 126, 128, 130, 132, 134 and 170. The amino acid and nucleotide sequences of exemplary 5T4 scFvs are presented below in Table 6.

Table 6. Amino acid and DNA sequences of illustrative 5T4 scFvs

Name DNA sequence SEQ ID NO: DNA sequences Amino acid sequence SEQ ID NO: amino acid sequence ALG.APV-178
ALG.APV-208
to 5T4 gaagtgcagctgctggagtccggaggaggactggtgcagcctggcggaagcctgaggctgagctgcgctgcctccggcttcacattcagcagctatgctatgagctgggtgaggcaagcccctggaaagggcctggagtgggtgtccgctatctccggcagcggcggaagcacctactacgctgactccgtcaagggcagg ttcaccatcagccgggacaacagcaagaacaccctgtacctgcagatgaatagcctcagggctgaagacaccgctgtgtactactgcgccaggtactatggcggctactactccgcctggatggactactggggacagggcacactggtgaccgtgtccagcggcggaggcggctccggaggcggtggctccggaggaggcggaagcggagggaggaggct ccgatattcagatgacacagtcccctagctccctgtccgccagcgtggggagatcgggtgaccatcacctgcagggccagccagtccatctccagctatttaaactggtaccagcagaagcctggaaaggctcccaagctgctgatctacgccgcttccagcctccagagcggcgtgcctagcaggttctccggctccggaagcggaacagact tcaccctgaccatcagctccctgcagcccgaggactccgctacctactactgccagcagacctatggctacctgcacaccttcggccagggcacaaagctggagatcaag 117 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVP SRFSGSGSGTDFTLTISSLQPEDSATYYCQQTYGYLHTFGQGTKLEIK 118 ALG.APV-179
ALG.APV.209
ALG.APV222
to 5T4 gaagtgcagctgctggagtccggaggaggactggtgcagcctggcggaagcctgaggctgagctgcgctgcctccggcttcacattcagcagctatgctatgagctgggtgaggcaagcccctggaaagggcctggagtgggtgtccgctatctccggcagcggcggaagcacctactacgctgactccgtcaagggcagg ttcaccatcagccgggacaacagcaagaacaccctgtacctgcagatgaatagcctcagggctgaagacaccgctgtgtactactgcgccaggtactatggcggctactactccgcctggatggactactggggacagggcacactggtgaccgtgtccagcggcggaggcggctccggaggcggtggctccggaggaggcggaagcggagggaggaggct ccgatattcagatgacacagtcccctagctccctgtccgccagcgtggggagatcgggtgaccatcacctgcagggccagccagtccatctccagcttcttaaactggtaccagcagaagcctggaaaggctcccaagctgctgatctacgccgcttccagcctccagagcggcgtgcctagcaggttctccggctccggaagcggaacag acttcaccctgaccatcagctccctgcagcccgaggactccgctacctactactgccagcagacctatggctacctgcacaccttcggccagggcacaaagctggagatcaag 119 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSFLNWYQQKPGKAPKLLIYAASSLQSGVP SRFSGSGSGTDFTLTISSLQPEDSATYYCQQTYGYLHTFGQGTKLEIK 120 ALG.APV-187
ALG.APV.210
ALG.APV223
to 5T4 gaagtgcagctgctggagtccggaggaggactggtgcagcctggcggaagcctgaggctgagctgcgctgcctccggcttcacattcagcagctatgctatgagctgggtgaggcaagcccctggaaagtgcctggagtgggtgtccgctatctccggcagcggcggaagcacctactacgctgactccgtcaagggca ggttcaccatcagccgggacaacagcaagaacaccctgtacctgcagatgaatagcctcagggctgaagacaccgctgtgtactactgcgccaggtactatggcggctactactccgcctggatggactactggggacagggcacactggtgaccgtgtccagcggcggaggcggctccggaggcggtggctccggaggcggaagcggagggaggagg ctccgatattcagatgacacagtcccctagctccctgtccgccagcgtggggagatcgggtgaccatcacctgcagggccagccagtccatctccagcttcttaaactggtaccagcagaagcctggaaaggctcccaagctgctgatctacgccgcttccagcctccagagcggcgtgcctagcaggttctccggctccggaagcggaac agacttcaccctgaccatcagctccctgcagcccgaggactccgctacctactactgccagcagacctatggctacctgcacaccttcggctgcggcacaaagctggagatcaag 121 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKCLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSFLNWYQQKPGKAPKLLIYAASSLQSGVP SRFSGSGSGTDFTLTISSLQPEDSATYYCQQTYGYLHTFGCGTKLEIK 122 ALG.APV-191
to 5T4 gaagtgcagctgctggagtccggaggaggactggtgcagcctggcggaagcctgaggctgagctgcgctgcctccggcttcacattcagcagctatgctatgagctgggtgaggcaagcccctggaaagtgcctggagtgggtgtccgctatctccggcagcggcggaagcacctactacgctgactccgtcaagggca ggttcaccatcagccgggacaacagcaagaacaccctgtacctgcagatgaatagcctcagggctgaagacaccgctgtgtactactgcgccaggtactatggcggctactactccgcctggatggactactggggacagggcacactggtgaccgtgtccagcggcggaggcggctccggaggcggtggctccggaggcggaagcggagggaggagg ctccgatattcagatgacacagtcccctagctccctgtccgccagcgtggggagatcgggtgaccatcacctgcagggccagccagtccatctccagcttcttaaactggtaccagcagaagcctggaaaggctcccaagctgctgatctacgccgcttccagcctccagagcggcgtgcctagcaggttctccggctccggaagcggaac agacttcaccctgaccatcagctccctgcagcccgaggacttcgctacctactactgccagcagacctatggctacctgcacaccttcggctgcggcacaaagctggagatcaag 123 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKCLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSFLNWYQQKPGKAPKLLIYAASSLQSGVP SRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYGYLHTFGCGTKLEIK 124 ALG.APV-196
to 5T4
ALG.APV-198
to 5T4 gaagtgcagctgctggagtccggaggaggactggtgcagcctggcggaagcctgaggctgagctgcgctgcctccggcttcgacttcgagagctatgctatgagctgggtgaggcaagcccctggaaagtgcctggagtgggtgtccgctatctccggcagcggcggaagcacctactacgctgactccgtcaaggg caggttcaccatcagccgggacaacagcaagaacaccctgtacctgcagatgaatagcctcagggctgaagacaccgctgtgtactactgcgccaggtactatggcggctactactccgcctggatggactactggggacagggcacactggtgaccgtgtccagcggcggaggcggctccggaggcggtggctccggaggcggaagcggagggagga ggctccgatattcagatgacacagtcccctagctccctgtccgccagcgtggggagatcgggtgaccatcacctgcagggccagccagtccatcaggagcgccctgaactggtaccagcagaagcctggaaaggctcccaagctgctgatctacgccgcttccagcctccagagcggcgtgcctagcaggttctccggctccggaagcgga acagacttcaccctgaccatcagctccctgcagcccgaggacttcgctacctactactgccagcagacctatggctacctgcacaccttcggctgcggcacaaagctggagatcaag 125 EVQLLESGGGLVQPGGSLRLSCAASGFDFESYAMSWVRQAPGKCLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSIRSALNWYQQKPGKAPKLLIYAASSLQSGVP SRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYGYLHTFGCGTKLEIK 126 ALG.APV-199
to 5T4 gaagtgcagctgctggagtccggaggaggactggtgcagcctggcggaagcctgaggctgagctgcgctgcctccggcttcgacttcgacagctatgctatgagctgggtgaggcaagcccctggaaagtgcctggagtgggtgtccgctatctccggcaggggcggaagcacctactacgctgactccgtcaagggca ggttcaccatcagccgggacaacagcaagaacaccctgtacctgcagatgaatagcctcagggctgaagacaccgctgtgtactactgcgccaggtactatggcggctactactccgcctggatggactactggggacagggcacactggtgaccgtgtccagcggcggaggcggctccggaggcggtggctccggaggcggaagcggagggaggagg ctccgatattcagatgacacagtcccctagctccctgtccgccagcgtggggagatcgggtgaccatcacctgcagggccagccagtccatcaggagcgccctgaactggtaccagcagaagcctggaaaggctcccaagctgctgatctacgccgcttccagcctccagagcggcgtgcctagcaggttctccggctccggaagcggaac agacttcaccctgaccatcagctccctgcagcccgaggacttcgctacctactactgccagcagacctatggctacctgcacaccttcggctgcggcacaaagctggagatcaag 127 EVQLLESGGGLVQPGGSLRLSCAASGFDFDSYAMSWVRQAPGKCLEWVSAISGRGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSIRSALNWYQQKPGKAPKLLIYAASSLQSGVP SRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYGYLHTFGCGTKLEIK 128 ALG.APV-006
to 5T4 gaggtgcagctgttggagagcgggggaggcttggtacagcctggggggtccctgcgcctctcctgtgcagccagcggattcacctttagcagctatgccatgagctgggtccgccaggctccaggaaggggctggagtgggtctcagctattagtggtagtggtggtagcacatactatgcagactccgtgaagggccggttcaccat ctcccgtgacaattccaagaacacgctgtatctgcaaatgaacagcctgcgtgccgaggacacggctgtatattattgtgcgcgctactacggtggttactactctgcttggatggactattggggccagggaaccctggtcaccgtctcctcaggcggtggaggcagcggtgggggtgggtctggaggcggtggcagtggcggcgga ggctctgacatccagatgacccagtctccatcctccctgagcgcatctgtaggagaccgcgtcaccatcacttgccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtttgcaaagtggggtcccatcacgtttcagtggcagtggaagcggga cagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttattactgtcaacagacttacggttacctgcacacttttggccaggggaccaagctggagatcaaa 129 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVP SRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYGYLHTFGQGTKLEIK 130 ALG.APV-010
to 5T4 gacatccagatgacccagtctccatcctccctgagcgcatctgtaggagaccgcgtcaccatcacttgccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtttgcaaagtggggtcccatcacgtttcagtggcagtggaagcgggaca gatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttattactgtcaacagacttacggttacctgcacacttttggccaggggaccaagctggagatcaaaggcggtggaggcagcggtgggggtgggtctggaggcggtggcagtggcggcggaggctctctgaggtgcagctgttggagagcgggggaggctt ggtacagcctggggggtccctgcgcctctcctgtgcagccagcggattcacctttagcagctatgccatgagctgggtccgccaggctccaggaaggggctggagtgggtctcagctattagtggtagtggtggtagcacatactatgcagactccgtgaagggccggttcaccatctcccgtgacaattccaagaacacgctgtatctgcaaat gaacagcctgcgtgccgaggacacggctgtatattattgtgcgcgctactacggtggttactactctgcttggatggactattggggccaggaaccctggtcaccgtctcctca 169 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYGYLHTFGQGTKLEIKGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFT ISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSS 170 ALG.APV-014
to 5T4 gaggtgcagctgttggagagcgggggaggcttggtacagcctggggggtccctgcgcctctcctgtgcagccagcggattcacctttagcagctatgccatgagctgggtccgccaggctccagggaagtgcctggagtgggtctcagctattagtggtagtggtggtagcacatactatgcagactccgtgaagggccggttcacca tctcccgtgacaattccaagaacacgctgtatctgcaaatgaacagcctgcgtgccgaggacacggctgtatattattgtgcgcgctactacggtggttactactctgcttggatggactattggggccagggaaccctggtcaccgtctcctcaggcggtggaggcagcggtgggggtgggtctggaggcggtggcagtggcggcgg aggctctgacatccagatgacccagtctccatcctccctgagcgcatctgtaggagaccgcgtcaccatcacttgccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtttgcaaagtggggtcccatcacgtttcagtggcagtggaagcgg gacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttattactgtcaacagacttacggttacctgcacacttttggctgcgggaccaagctggagatcaaa 131 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKCLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVP SRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYGYLHTFGCGTKLEIK 132 ALG.APV-018
to 5T4 gacatccagatgacccagtctccatcctccctgagcgcatctgtaggagaccgcgtcaccatcacttgccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtttgcaaagtggggtcccatcacgtttcagtggcagtggaagcgggaca gatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttattactgtcaacagacttacggttacctgcacacttttggctgcgggaccaagctggagatcaaaggcggtggaggcagcggtgggggtgggtctggaggcggtggcagtggcggcggaggctctctgaggtgcagctgttggagagcgggggaggct tggtacagcctggggggtccctgcgcctctcctgtgcagccagcggattcacctttagcagctatgccatgagctgggtccgccaggctccagggaagtgcctggagtgggtctcagctattagtggtagtggtggtagcacatactatgcagactccgtgaagggccggttcaccatctcccgtgacaattccaagaacacgctgtatctgca aatgaacagcctgcgtgccgaggacacggctgtatattattgtgcgcgctactacggtggttactactctgcttggatggactattggggccagggaaccctggtcaccgtctcctca 133 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYGYLHTFGCGTKLEIKGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKCLEWVSAISGSGGSTYYADSVKGRFTIS RDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSS 134

[00196] In some embodiments, the 5T4 binding polypeptide comprises, in order from amino terminus to carboxy terminus (or in order from carboxy terminus to amino terminus), (i) a 5T4 binding domain, (ii) a hinge region, (iii) an immunoglobulin constant region, ( iv) a carboxy-terminal linker (or amino-terminal linker); and (v) a second binding domain. In additional embodiments, the second binding domain is a 4-1BB-specific scFv. In some embodiments, the 5T4 binding polypeptide comprises, in order from amino terminus to carboxy terminus (or in order from carboxy terminus to amino terminus), (i) a second binding domain, (ii) a hinge region, (iii) an immunoglobulin constant region, (iv) a carboxy terminal linker (or amino-terminal linker) and (v) 5T4-binding domain. In additional embodiments, the second binding domain comprises or is a 4-1BB binding domain. In certain embodiments, the 5T4 binding protein may comprise an effector cell binding domain for recruiting the effector to target cells expressing 5T4. In certain embodiments, the effector cell binding domain specifically binds to 4-1BB.

[00197] In some embodiments, the second binding domain of a 5T4 binding polypeptide described herein is a 4-1BB binding domain and comprises one or more 4-1BB binding sequences (e.g., CDRs or variable regions) disclosed in publication of PCT application No. WO 2016/185016; PCT Application No. PCT/EP2017/059656; Dubrot et al. , 2010; Gauttier et al. , 2014; Kim et al. , 2001; McMillin et al. , 2006; Melero et al. , 1997; Miller et al. , 2002; Sallin et al. , 2014; Taraban et al. , 2002; Uno et al. , 2006; Vinay and Kwon, 2012; Wilcox et al. , 2002, each of which is incorporated herein by reference in its entirety.

[00198] In some embodiments, the second binding domain specifically binds 4-1BB and comprises an immunoglobulin light chain variable region (V _L ) and an immunoglobulin heavy chain variable region (V _H ); wherein V _L contains an amino acid sequence that is at least about 93% identical, at least about 95% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical identical or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 16 and 22; and wherein V _H contains an amino acid sequence that is at least about 93% identical, at least about 95% identical, at least about 97% identical, at least about 98% identical, at least about 99 % identical or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 20, 26 and 28, where the polypeptide is a polyspecific polypeptide in the scFv-Fc-scFv format and contains a Y to H substitution in HCDR1 in position 150 V _H and/or replacing R with H in FR3 at position 127 V _H. In some embodiments, the second binding domain specifically binds 4-1BB and contains V _L and V _H ; where V _L contains an amino acid sequence selected from the group consisting of SEQ ID NO: 16 and 22; and wherein V _H contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 20, 26 and 28.

[00199] In some embodiments, the second binding domain is a 4-1BB binding domain polypeptide, wherein the polypeptide is a scFv containing at least about 82% sequence, at least about 85% sequence, at least about by 87%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95 %, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO : 110, 112, 114 and 116. Exemplary 4-1BB binding domain sequences are presented in Tables 6 and 7 and discussed below.

4T1-binding domains and proteins containing them

[00200] In some embodiments, the present invention provides 4-1BB binding domains that specifically bind to 4-1BB (eg, human 4-1BB). 4-1BB is also known as CD137 or TNFRSF9 and is a member of the tumor necrosis factor (TNF) receptor family. 4-1BB is expressed on multiple cell types, including activated T cell subsets (eg, CD4+ and CD8+ T cells and regulatory T cells (Tregs)), natural killer (NK) cells, dendritic cells, monocytes, mast cells, and eosinophils. Activation of 4-1BB on CD8+ T cells maintains and/or enhances cell activation, whereas activation of 4-1BB on CD4+ T cells can initiate cell activation and, in some cases, lead to activation-induced cell death. Activation of 4-1BB on Tregs generally suppresses Tregs function. Several studies have demonstrated the induction of tumor immunity by the use of 4-1BB antagonist antibodies. Exemplary nucleotide and amino acid sequences of human 4-1BB are provided in SEQ ID NOs: 163, 167 and SEQ ID NOs: 164 and 166, respectively.

[00201] In some embodiments, the present invention further provides polypeptides comprising a 4-1BB binding domain (eg, 4-1BB binding polypeptides). In certain variations, the 4-1BB binding polypeptide contains a hinge region that is carboxy-terminal to the 4-1BB binding domain and an immunoglobulin constant region. In further variations, the 4-1BB binding polypeptide comprises a carboxy-terminal binding domain, a linker carboxy-terminal to the immunoglobulin constant region, and a second binding domain carboxy-terminal to the carboxy-terminal linker.

[00202] In other variations, the 4-1BB binding polypeptide comprises a hinge region amino-terminal to the polypeptide containing the 4-1BB binding domain and an immunoglobulin constant region amino-terminal to the hinge region.

[00203] In some embodiments, the 4-1BB binding domains described herein bind an epitope located on the extracellular domain of 4-1BB (eg, the epitope contained in SEQ ID NO: 166). In certain aspects, the epitope is discontinuous and/or conformational.

[00204] The 4-1BB binding domain polypeptide can specifically bind to human 4-1BB and comprises heavy chain CDR1 (HCDR1), HCDR2, HCDR3, light chain CDR1 (LCDR1), LCDR2 and LCDR3, where HCDR1 contains an amino acid sequence selected from group consisting of SEQ ID NO: 2, 18, 24; HCDR2 contains the amino acid sequence SEQ ID NO: 4; HCDR3 contains the amino acid sequence SEQ ID NO: 6; LCDR1 contains the amino acid sequence SEQ ID NO: 8; LCDR2 contains the amino acid sequence SEQ ID NO: 10; and LCDR3 contains the amino acid sequence SEQ ID NO: 12, where the specified polypeptide is a polyspecific polypeptide in the scFv-Fc-scFv format and contains a Y to H substitution in HCDR1 at position 150 V _H to 4-1BB and/or an R to H substitution at FR3 in position 127 V _H to 4-1BB.

[00205] In particular embodiments, the 4-1BB binding domain polypeptide may specifically bind to human 4-1BB and comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein (a) HCDR1 contains the amino acid sequence set forth in SEQ ID NO : 2; (b) HCDR2 contains the amino acid sequence shown in SEQ ID NO: 4; (c) HCDR3 contains the amino acid sequence shown in SEQ ID NO: 6; (d) LCDR1 contains the amino acid sequence shown in SEQ ID NO: 8; LCDR2 contains the amino acid sequence shown in SEQ ID NO: 10; and (f) LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 12. In other particular embodiments, the 5T4 binding domain polypeptide specifically binds to human 5T4 and comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein (a) HCDR1 contains the amino acid sequence shown in SEQ ID NO: 18; (b) HCDR2 contains the amino acid sequence shown in SEQ ID NO: 4; (c) HCDR3 contains the amino acid sequence shown in SEQ ID NO: 6; (d) LCDR1 contains the amino acid sequence shown in SEQ ID NO: 8; (e) LCDR2 contains the amino acid sequence set forth in SEQ ID NO: 10, and (f) LCDR3 contains the amino acid sequence set forth in SEQ ID NO: 12. In other particular embodiments, the 5T4 binding domain polypeptide specifically binds to human 5T4 and comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, where (a) HCDR1 contains the amino acid sequence presented in SEQ ID NO: 24; (b) HCDR2 contains the amino acid sequence shown in SEQ ID NO: 4; (c) HCDR3 contains the amino acid sequence shown in SEQ ID NO: 6; (d) LCDR1 contains the amino acid sequence shown in SEQ ID NO: 8; (e) LCDR2 contains the amino acid sequence shown in SEQ ID NO: 10; and (f) LCDR3 contains the amino acid sequence set forth in SEQ ID NO: 12.

[00206] In certain embodiments, the 4-1BB binding domain polypeptide comprises a light chain variable region (VL) amino acid sequence selected from the group consisting of SEQ ID NOs: 16 and 22. In certain embodiments, the 4-1BB binding domain polypeptide contains an amino acid sequence of at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94 %, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5 % or 100% identical to the amino acid sequence of a heavy chain variable region ( _VH ) selected from the group consisting of SEQ ID NO: 14, 20, 26 and 28, wherein the polypeptide is a polyspecific polypeptide in the format scFv-Fc-scFv and contains a Y to H substitution in HCDR1 at position 150 V _H to 4-1BB and/or an R to H substitution in FR3 at position 127 V _H to 4-1BB. In certain embodiments, the 4-1BB binding domain polypeptide comprises a heavy chain variable region (VH) amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 20, 26, and 28.

[00207] In certain embodiments, the 4-1BB binding domain polypeptide comprises a V _H region containing the amino acid sequence of SEQ ID NO: 14 and a V _L region containing the amino acid sequence of SEQ ID NO: 16. In certain embodiments, the 4-1BB polypeptide -binding domain comprises a V _H region containing the amino acid sequence of SEQ ID NO: 20 and a V _L region containing the amino acid sequence of SEQ ID NO: 22. In certain embodiments, the 4-1BB binding domain polypeptide comprises a V _H region containing the amino acid sequence SEQ ID NO: 26, and a V _L region containing the amino acid sequence of SEQ ID NO: 16. In certain embodiments, the 4-1BB binding domain polypeptide comprises a V _H region containing the amino acid sequence of SEQ ID NO: 28, and a V _L region. containing the amino acid sequence SEQ ID NO: 16.

[00208] Exemplary 4-1BB binding domain sequences are presented in Table 7.

Table 7. Exemplary polypeptide and nucleotide sequences of the 4-1BB binding domain

Design Component DNA sequence SEQ ID NO: DNA sequences Amino acid sequence SEQ ID NO: amino acid sequence ALG.APV-178
ALG.APV-179
ALG.APV-187
ALG.APV-198
ALG.APV-199
ALG.APV-208
ALG.APV-209
ALG.APV-210
ALG.APV-222
ALG.APV-223 HCDR1 ggattcacctttctcacggttct 1 GFTFSHGS 2 HCDR2 atttcttctggttctggttctaca 3 ISSGSGST 4 HCDR3 gcgcgctcttcttactacggttcttactactctattgactat 5 ARSSYYGSYYSIDY 6 LCDR1 cagagcattagcagctat 7 QSISSY 8 LCDR2 gctgcatcc 9 A.A.S. 10 LCDR3 caacagtactacgacaacctgcccact eleven QQYYDNLPT 12 V _H gaggtgcagctgttggagagcgggggaggcttggtacagcctggggggtccctgcgcctctctcctgtgcagccagcggattcaccttttctcacggttctatgtactgggtccgccaggctccagggaaggggctggagtgggtctcatctatttcttctggttctggttctacatactatgcagactccgtgaagggccggt tcaccatctcccatgacaattccaagaacacgctgtatctgcaaatgaacagcctgcgtgccgaggacacggctgtatattattgtgcgcgctcttcttactacggttcttactactctattgactattggggccagggaaccctggtcaccgtctcctca 13 EVQLLESGGGLVQPGGSLRLSCAASGFTFSHGSMYWVRQAPGKGLEWVSSISSGSGSTYYADSVKGRFTISHDNSKNTLYLQMNSLRAEDTAVYYCARSSYYGSYYSIDYWGQGTLVTVSS 14 V _L gacatccagatgacccagtctccatcctccctgagcgcatctgtaggagaccgcgtcaccatcacttgccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtttgcaaagtggggtcccatcacgtttcagtggcagtggaagcgggaca gatttcactctcaccatcagcagtctacaacctgaagattttgcaacttattactgtcaacagtactacgacaacctgcccacttttggccaggggaccaagctggagatcaaa 15 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYDNLPTFGQGTKLEIK 16 ALG.APV-191 HCDR1 ggattcacctttgattacggttct 17 GFTFDYGS 18 HCDR2 atttcttctggttctggttctaca 3 ISSGSGST 4 HCDR3 gcgcgctcttcttactacggttcttactactctattgactat 5 ARSSYYGSYYSIDY 6 LCDR1 cagagcattagcagctat 7 QSISSY 8 LCDR2 gctgcatcc 9 A.A.S. 10 LCDR3 caacagtactacgacaacctgcccact eleven QQYYDNLPT 12 V _H gaggtgcagctgttggagagcgggggaggcttggtacagcctggggggtccctgcgcctctctcctgtgcagccagcggattcacctttgattacggttctatgtactgggtccgccaggctccaggaaggggctggagtgggtctcatctatttcttctggttctggttctacatactatgcagactccgtgaagggccggtt caccatctcccacgacaattccaagaacacgctgtatctgcaaatgaacagcctgcgtgccgaggacacggctgtatattattgtgcgcgctcttcttactacggttcttactactctattgactattggggccagggaaccctggtcaccgtctcctca 19 EVQLLESGGGLVQPGGSLRLSCAASGFTFDYGSMYWVRQAPGKGLEWVSSISSGSGSTYYADSVKGRFTISHDNSKNTLYLQMNSLRAEDTAVYYCARSSYYGSYYSIDYWGQGTLVTVSS 20 V _L gacatccagatgacccagtctccatcctccctgagcgcatctgtaggagaccgcgtcaccatcacttgccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtttgcacagtggggtcccatcacgtttcagtggcagtggaagcgggaca gatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttattactgtcaacagtactacgacaacctgcccacttttggccaggggaccaagctggagatcaaa 21 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYDNLPTFGQGTKLEIK 22 ALG.APV-196 HCDR1 ggattcacctttcttacggttct 23 GFTFSYGS 24 HCDR2 atttcttctggttctggttctaca 3 ISSGSGST 4 HCDR3 gcgcgctcttcttactacggttcttactactctattgactat 5 ARSSYYGSYYSIDY 6 LCDR1 cagagcattagcagctat 7 QSISSY 8 LCDR2 gctgcatcc 9 A.A.S. 10 LCDR3 caacagtactacgacaacctgcccact eleven QQYYDNLPT 12 V _H gaggtgcagctgttggagagcgggggaggcttggtacagcctggggggtccctgcgcctctctcctgtgcagccagcggattcacctttctcttacggttctatgtactgggtccgccaggctccagggaaggggctggagtgggtctcatctatttcttctggttctggttctacatactatgcagactccgtgaagggccggt tcaccatctcccatgacaattccaagaacacgctgtatctgcaaatgaacagcctgcgtgccgaggacacggctgtatattattgtgcgcgctcttcttactacggttcttactactctattgactattggggccagggaaccctggtcaccgtctcctca 25 EVQLLESGGGLVQPGGSLRLSCAASGFTFSYGSMYWVRQAPGKGLEWVSSISSGSGSTYYADSVKGRFTISHDNSKNTLYLQMNSLRAEDTAVYYCARSSYYGSYYSIDYWGQGTLVTVSS 26 V _L gacatccagatgacccagtctccatcctccctgagcgcatctgtaggagaccgcgtcaccatcacttgccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtttgcaaagtggggtcccatcacgtttcagtggcagtggaagcgggaca gatttcactctcaccatcagcagtctacaacctgaagattttgcaacttattactgtcaacagtactacgacaacctgcccacttttggccaggggaccaagctggagatcaaa 15 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYDNLPTFGQGTKLEIK 16 ALG.APV-004
ALG.APV-006
ALG.APV-010
ALG.APV-014
ALG.APV-018 HCDR1 ggattcacctttcttacggttct 23 GFTFSYGS 24 HCDR2 atttcttctggttctggttctaca 3 ISSGSGST 4 HCDR3 gcgcgctcttcttactacggttcttactactctattgactat 5 ARSSYYGSYYSIDY 6 LCDR1 cagagcattagcagctat 7 QSISSY 8 LCDR2 gctgcatcc 9 A.A.S. 10 LCDR3 caacagtactacgacaacctgcccact eleven QQYYDNLPT 12 V _H gaggtgcagctgttggagagcgggggaggcttggtacagcctggggggtccctgcgcctctctcctgtgcagccagcggattcacctttctcttacggttctatgtactgggtccgccaggctccagggaaggggctggagtgggtctcatctatttcttctggttctggttctacatactatgcagactccgtgaagggccggt tcaccatctcccgtgacaattccaagaacacgctgtatctgcaaatgaacagcctgcgtgccgaggacacggctgtatattattgtgcgcgctcttcttactacggttcttactactctattgactattggggccagggaaccctggtcaccgtctcctca 27 EVQLLESGGGLVQPGGSLRLSCAASGFTFSYGSMYWVRQAPGKGLEWVSSISSGSGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSSYYGSYYSIDYWGQGTLVTVSS 28 V _L gacatccagatgacccagtctccatcctccctgagcgcatctgtaggagaccgcgtcaccatcacttgccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtttgcaaagtggggtcccatcacgtttcagtggcagtggaagcgggaca gatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttattactgtcaacagtactacgacaacctgcccacttttggccaggggaccaagctggagatcaaa 15 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYDNLPTFGQGTKLEIK 16

[00209] In some embodiments, the present invention provides polyspecific polypeptides (eg, bispecific polypeptides) containing a 4-1BB binding domain. A polyspecific 4-1BB binding polypeptide or protein may contain two binding domains (the domains may be designed to specifically bind to the same or different targets), a hinge region, a linker (eg, a carboxy-terminal or amino-terminal linker), and an immunoglobulin constant region. The polyspecific 4-1BB binding protein may be a homodimeric protein containing two identical polypeptides linked by a disulfide bond. In some embodiments, the 4-1BB binding domains can be derived from a 4-1BB binding monoclonal antibody (e.g., urelumab (BMS-66513) or PF-05082566 (Pfizer)) derived from the 4-1BB binding domain described in PCT Application Publication No. WO 2016/185016, or derived from the 4-1BB binding domain according to PCT Application No. PCT/EP2017/059656.

[00210] In some embodiments, a polyspecific polypeptide comprising a 4-1BB binding domain may comprise, in order from amino terminus to carboxy terminus: (i) a first binding domain, (ii) a hinge region, (iii) an immunoglobulin constant region, (iv) ) linker binding domains and (v) 4-1BB binding domain. In some embodiments, the polyspecific polypeptide comprising a 4-1BB binding domain may comprise, in order from amino terminus to carboxy terminus: (i) a 4-1BB binding domain, (ii) a linker binding domains, (iii) an immunoglobulin constant region, ( iv) a hinge region; and (v) a first binding domain. The amino acid and nucleotide sequences for exemplary 4-1BB binding domain sequences are presented in Table 7.

[00211] In certain embodiments, the 4-1BB binding protein or polypeptide may comprise one or more additional binding domains (eg, a second binding domain) that bind a target other than 4-1BB. These other binding domains may contain, for example, a particular cytokine or molecule that targets the binding domain polypeptide, for example, to a specific cell type, toxin, additional cellular receptor or antibody.

[00212] In some embodiments, the 4-1BB-binding polypeptide is capable of forming a heterodimer with a second polypeptide chain and contains a hinge region (a) immediately amino-terminal to an immunoglobulin constant region (e.g., amino-terminal to a CH2 domain if the immunoglobulin constant region includes domains CH2 and CH3, or amino-terminal to the CH3 domain if the immunoglobulin subregions include CH3 and CH4 domains), (b) located between and connecting the binding domain (e.g., scFv) and the immunoglobulin heterodimerization domain, (c) located between the immunoglobulin heterodimerization domain and an immunoglobulin constant region (for example, when the immunoglobulin constant region includes the CH2 and CH3 domains or the CH3 and CH4 domains), and connecting them, (d) located between and connecting the immunoglobulin constant region and the binding domain, (e) at the amino terminus of the polypeptide chain or (f) at the carboxy terminus of a polypeptide chain. A polypeptide chain containing a hinge region as described herein will be capable of binding to another polypeptide chain to form a heterodimeric protein as provided herein, and the resulting heterodimer will contain a binding domain retaining its target specificity or its specific target binding affinity .

[00213] In certain embodiments, the 4-1BB binding polypeptide may comprise a target cell binding domain for recruiting target cells to effector cells expressing 4-1BB. In certain embodiments, the target cell binding domain specifically binds to 5T4. In certain embodiments, a 4-1BB binding protein as described herein may comprise (i) a binding domain that specifically binds 4-1BB, and (ii) another binding domain that specifically binds 5T4. Non-limiting examples of anti-5T4 antibodies from which the 5T4 binding domain can be derived include those described in PCT Application Publication No. WO 2016/185016 and PCT Application No. PCT/EP2017/059656.

[00214] In some embodiments, the 4-1BB binding polypeptide provided herein is a polypeptide comprising two scFvs. In some embodiments, the two scFvs contain 4-1BB binding domains. In certain embodiments, the two scFvs contain identical 4-1BB binding domains. In other embodiments, the two scFvs contain different 4-1BB binding domains. In other embodiments, the polypeptide comprises a 4-1BB binding domain as a first scFv and a target cell binding domain as a second scFv. For example, the target cell binding domain may be a 5T4-specific scFv.

[00215] In some embodiments, the bispecific 4-1BB binding polypeptides and proteins provided herein comprise at least about 82% anti-4-1BB scFv, at least about 85%, at least about 85% anti-4-1BB scFv. 87%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95% is at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 110, 112, 114 and 116, where the specified polypeptide is a polyspecific polypeptide in the scFv-Fc-scFv format and contains a Y to H substitution in HCDR1 at position 150 V _H to 4-1BB and/or an R to H substitution in FR3 at position 127 V _H to 4-1BB. In certain embodiments, the bispecific 4-1BB binding polypeptides and proteins provided herein comprise an anti-4-1BB scFv comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 110, 112, 114, and 116. Amino acid and The nucleotide sequences of exemplary 4-1BB scFvs are presented below in Table 8.

Table 8. Amino acid and DNA sequences of exemplary scFvs for 4-1BB

Design DNA sequence SEQ ID NO: DNA sequences Amino acid sequence SEQ ID NO: amino acid sequence ALG.APV-178-4-1BB
ALG.APV-179-4-1BB
ALG.APV-187-4-1BB
ALG.APV-198-4-1BB
ALG.APV-199-4-1BB
ALG.APV-208-4-1BB
ALG.APV-209-4-1BB
ALG.APV-210-4-1BB
ALG.APV-222-4-1BB
ALG.APV-223-4-1BB gaggtgcagctgttggagagcgggggaggcttggtacagcctggggggtccctgcgcctctctcctgtgcagccagcggattcaccttttctcacggttctatgtactgggtccgccaggctccagggaaggggctggagtgggtctcatctatttcttctggttctggttctacatactatgcagactccgtgaagggccggt tcaccatctcccatgacaattccaagaacacgctgtatctgcaaatgaacagcctgcgtgccgaggacacggctgtatattattgtgcgcgctcttcttactacggttcttactactctattgactattggggccagggaaccctggtcaccgtctcctcaggtggaggtggctccgggggtggaggttccggaggaggcggatcaggtggaggcgg aagcgacatccagatgacccagtctccatcctccctgagcgcatctgtaggagaccgcgtcaccatcacttgccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaaccaggggaaagcccctaagctcctgatctatgctgcatccagtttgcaaagtggggtcccatcacgtttcagtggcagtggaagcggga cagatttcactctcaccatcagcagtctacaacctgaagattttgcaacttattactgtcaacagtactacgacaacctgcccacttttggccaggggaccaagctggagatcaaa 109 EVQLLESGGGLVQPGGSLRLSCAASGFTFSHGSMYWVRQAPGKGLEWVSSISSGSGSTYYADSVKGRFTISHDNSKNTLYLQMNSLRAEDTAVYYCARSSYYGSYYSIDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQYYDNLPTFGQGTKLEIK 110 ALG.APV-191-4-1BB gaggtgcagctgttggagagcgggggaggcttggtacagcctggggggtccctgcgcctctctcctgtgcagccagcggattcacctttgattacggttctatgtactgggtccgccaggctccaggaaggggctggagtgggtctcatctatttcttctggttctggttctacatactatgcagactccgtgaagggccggtt caccatctcccacgacaattccaagaacacgctgtatctgcaaatgaacagcctgcgtgccgaggacacggctgtatattattgtgcgcgctcttcttactacggttcttactactctattgactattggggccagggaaccctggtcaccgtctcctcaggtggaggtggctccgggggtggaggttccggaggaggcggatcaggtggaggcgg aagcgacatccagatgacccagtctccatcctccctgagcgcatctgtaggagaccgcgtcaccatcacttgccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaaccaggggaaagcccctaagctcctgatctatgctgcatccagtttgcacagtggggtcccatcacgtttcagtggcagtggaagcggga cagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttattactgtcaacagtactacgacaacctgcccacttttggccaggggaccaagctggagatcaaa 111 evqllesggglvqpggslrlscaasgftfdygsmywvrqapgkglewvssissgsgstyyadsvkgrftishdnskntlylqmnslraedtavyycarssyygsyysidywgqgtlvtvssggggsggggsggggsggggsdiqmtqspsslsasvgdrvtitcrasqsissylnwyqqk pgkapklliyaasslhsgvpsrfsgsgsgtdftltisslqpedfatyycqqyydnlptfgqgtkleik 112 ALG.APV-196-4-1BB gaggtgcagctgttggagagcgggggaggcttggtacagcctggggggtccctgcgcctctctcctgtgcagccagcggattcacctttctcttacggttctatgtactgggtccgccaggctccagggaaggggctggagtgggtctcatctatttcttctggttctggttctacatactatgcagactccgtgaagggccggt tcaccatctcccatgacaattccaagaacacgctgtatctgcaaatgaacagcctgcgtgccgaggacacggctgtatattattgtgcgcgctcttcttactacggttcttactactctattgactattggggccagggaaccctggtcaccgtctcctcaggtggaggtggctccgggggtggaggttccggaggaggcggatcaggtggaggcgg aagcgacatccagatgacccagtctccatcctccctgagcgcatctgtaggagaccgcgtcaccatcacttgccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaaccaggggaaagcccctaagctcctgatctatgctgcatccagtttgcaaagtggggtcccatcacgtttcagtggcagtggaagcggga cagatttcactctcaccatcagcagtctacaacctgaagattttgcaacttattactgtcaacagtactacgacaacctgcccacttttggccaggggaccaagctggagatcaaa 113 EVQLLESGGGLVQPGGSLRLSCAASGFTFSYGSMYWVRQAPGKGLEWVSSISSGSGSTYY
ADSVKGRFTISHDNSKNTLYLQMNSLRAEDTAVYYCARSSYYGSYYSIDYWGQGTLVTVS
SGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQK
PGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYDNLPTFG
QGTKLEIK 114 ALG.APV-006-4-1BB
ALG.APV-010-4-1BB
ALG.APV-014-4-1BB
ALG.APV-018-4-1BB gaggtgcagctgttggagagcgggggaggcttggtacagcctggggggtccctgcgcctctctcctgtgcagccagcggattcacctttctcttacggttctatgtactgggtccgccaggctccagggaaggggctggagtgggtctcatctatttcttctggttctggttctacatactatgcagactccgtgaagggccggt tcaccatctcccgtgacaattccaagaacacgctgtatctgcaaatgaacagcctgcgtgccgaggacacggctgtatattattgtgcgcgctcttcttactacggttcttactactctattgactattggggccagggaaccctggtcaccgtctcctcaggcggcggcggcagcggcggcggcggcagcggcggcggaggctccggcgg cggcggcagcgacatccagatgacccagtctccatcctccctgagcgcatctgtaggagaccgcgtcaccatcacttgccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtttgcaaagtggggtcccatcacgtttcagtggcagtgga agcgggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttattactgtcaacagtactacgacaacctgcccacttttggccaggggaccaagctggagatcaaa 115 evqllesggglvqpggslrlscaasgftfsygsmywvrqapgkglewvssissgsgstyyadsvkgrftisrdnskntlylqmnslraedtavyycarssyygsyysidywgqgtlvtvssggggsggggsggggsggggsdiqmtqspsslsasvgdrvtitcrasqsissylnwyqqk pgkapklliyaasslqsgvpsrfsgsgsgtdftltisslqpedfatyycqqyydnlptfgqgtkleik 116

Bispecific molecules

[00216] In some embodiments, the present invention provides polyspecific 5T4-binding polypeptides comprising two binding domains, where one of the binding domains is specific for 5T4 and the second binding domain is specific for another target antigen. In some embodiments, the present invention provides bispecific 4-1BB binding polypeptides comprising two binding domains, wherein the binding domain is specific for 4-1BB and the second binding domain is specific for another target antigen. In particular embodiments, the present invention relates to bispecific polypeptides containing two binding domains, where one of the binding domains is specific for 4-1BB and the second binding domain is specific for 5T4. Such embodiments are referred to herein as anti-5T4 x 4-1BB molecules or anti-5T4 x 4-1BB polypeptides or proteins. In particular embodiments, the 5T4 and 4-1BB binding domains of the 5T4 x 4-1BB molecule are scFv domains that specifically bind to 5T4 and 4-1BB, respectively.

[00217] In some embodiments, the 5T4 x 4-1BB molecules described herein comprise, in order from amino terminus to carboxy terminus, (i) a 5T4 binding domain; (ii) linker binding domains; and (iii) 4-1BB binding domain. In some embodiments, the 5T4 x 4-1BB molecules described herein comprise, in order from amino terminus to carboxy terminus, (i) a 4-1BB binding domain; (ii) linker binding domains; and (iii) 5T4-binding domain. In some embodiments, the 5T4 x 4-1BB molecules described herein contain, in order from amino terminus to carboxy terminus (or in order from carboxy terminus to amino terminus), (i) a 5T4 binding domain, (ii) a hinge region (iii) ) an immunoglobulin constant region, (iv) a carboxy-terminal linker (or amino-terminal linker), and (v) a second binding domain.

[00218] The 5T4 x 4-1BB molecules described herein may contain any combination of the 5T4 and 4-1BB binding domain sequences presented in Tables 4-7. In particular embodiments, the 5T4 x 4-1BB molecules of the present invention comprise a 5T4 binding domain and a 4-1BB binding domain, each comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3. In some embodiments, the 5T4 x 4-1BB molecules comprise (a) a first scFv domain comprising: (i) a VH comprising an HCDR1 amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 52, and 60, amino acid sequence HCDR2 selected from the group consisting of SEQ ID NO: 32 and 62, and the amino acid sequence of HCDR3, which is SEQ ID NO: 34; and (ii) VL comprising an LCDR1 amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 42 and 54, an LCDR2 amino acid sequence represented by SEQ ID NO: 10, and an LCDR3 amino acid sequence represented by SEQ ID NO: 36; and (b) a second scFv domain comprising (i) a VH comprising an HCDR1 amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18 and 24, an HCDR2 amino acid sequence represented by SEQ ID NO: 4, and an the HCDR3 sequence represented by SEQ ID NO: 6; and (ii) VL comprising the LCDR1 amino acid sequence of SEQ ID NO: 8, the LCDR2 amino acid sequence of SEQ ID NO: 10, and the LCDR3 amino acid sequence of SEQ ID NO: 12.

[00219] In some embodiments, the 5T4 x 4-1BB molecules of the present invention comprise a 5T4 binding domain and a 4-1BB binding domain, each comprising a V _H and a V _L domain. In some embodiments, the 5T4 x 4-1BB molecules comprise a first scFv domain comprising i) a V _H containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 38, 46, 56 and 64, and a V _L containing an amino acid sequence selected from the group consisting of SEQ ID NO: 40, 44, 48, 50, 58, 66, 68 and 70; and a second scFv domain comprising ii) V _H containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 20, 26 and 28, and V _L containing an amino acid sequence selected from the group consisting of SEQ ID NO: 16 and 22.

[00220] In certain embodiments, the 5T4 binding domain of the molecule to 5T4 x 4-1BB contains or is at least about 90% scFv, at least about 91%, at least about 92%, at least at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 118, 120, 122, 124, 126, 128, 130, 132, 134 and 170 , where the specified molecule is a polyspecific polypeptide in the scFv-Fc-scFv format and contains a Y to F substitution in LCDR1 at position 99 V _L to 5T4 and/or an F to S substitution in FR3 at position 148 V _L to 5T4. In some embodiments, the 5T4 binding domain of the 5T4 x 4-1BB molecule comprises a scFv comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 118, 120, 122, 124, 126, 128, 130, 132, 134, and 170.

[00221] In certain embodiments, the 4-1BB binding domain of the 5T4 x 4-1BB molecule contains or is at least about 90% scFv, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 110, 112, 114, and 116, wherein said molecule is a polyspecific polypeptide in the format scFv-Fc-scFv and contains a Y to H substitution in HCDR1 at position 150 V _H to 4-1BB and/or an R to H substitution in FR3 at position 127 V _H to 4-1BB. In certain embodiments, the 4-1BB binding domain of the 5T4 x 4-1BB molecule comprises a scFv containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 110, 112, 114, and 116.

[00222] In particular embodiments, the 5T4 x 4-1BB molecule contains a combination of CDR sequences, V _H /V _L sequences and/or scFv sequences, as presented below in Table 9.

Table 9. Exemplary combinations of 5T4 and 4-1BB binding sequences

ALG.APV-178 (scFv-Fc-scFv format) Binding domain to 4-1BB Binding domain to 5T4 Component SEQ ID NO: Component SEQ ID NO: HCDR1 2 HCDR1 thirty HCDR2 4 HCDR2 32 HCDR3 6 HCDR3 34 LCDR1 8 LCDR1 8 LCDR2 10 LCDR2 10 LCDR3 12 LCDR3 36 V _H 14 V _H 38 V _L 16 V _L 40 scFv 110 scFv 118 ALG.APV-179, ALG.APV-209, ALG.APV-222 (scFv-Fc-scFv format) Binding domain to 4-1BB Binding domain to 5T4 Component SEQ ID NO: Component SEQ ID NO: HCDR1 2 HCDR1 thirty HCDR2 4 HCDR2 32 HCDR3 6 HCDR3 34 LCDR1 8 LCDR1 42 LCDR2 10 LCDR2 10 LCDR3 12 LCDR3 36 V _H 14 V _H 38 V _L 16 V _L 44 scFv 110 scFv 120 ALG.APV-187, ALG.APV-210, ALG.APV-223 (scFv-Fc-scFv format) Binding domain to 4-1BB Binding domain to 5T4 Component SEQ ID NO: Component SEQ ID NO: HCDR1 2 HCDR1 thirty HCDR2 4 HCDR2 32 HCDR3 6 HCDR3 34 LCDR1 8 LCDR1 42 LCDR2 10 LCDR2 10 LCDR3 12 LCDR3 36 V _H 14 V _H 46 V _L 16 V _L 48 scFv 110 scFv 122 ALG.APV-191 (scFv-Fc-scFv format) Binding domain to 4-1BB Binding domain to 5T4 Component SEQ ID NO: Component SEQ ID NO: HCDR1 18 HCDR1 thirty HCDR2 4 HCDR2 32 HCDR3 6 HCDR3 34 LCDR1 8 LCDR1 42 LCDR2 10 LCDR2 10 LCDR3 12 LCDR3 36 V _H 20 V _H 46 V _L 22 V _L 50 scFv 112 scFv 124 ALG.APV-196 (scFv-Fc-scFv format) Binding domain to 4-1BB Binding domain to 5T4 Component SEQ ID NO: Component SEQ ID NO: HCDR1 24 HCDR1 52 HCDR2 4 HCDR2 32 HCDR3 6 HCDR3 34 LCDR1 8 LCDR1 54 LCDR2 10 LCDR2 10 LCDR3 12 LCDR3 36 V _H 26 V _H 56 V _L 16 V _L 58 scFv 114 scFv 126 ALG.APV-198 (scFv-Fc-scFv format) Binding domain to 4-1BB Binding domain to 5T4 Component SEQ ID NO: Component SEQ ID NO: HCDR1 2 HCDR1 52 HCDR2 4 HCDR2 32 HCDR3 6 HCDR3 34 LCDR1 8 LCDR1 54 LCDR2 10 LCDR2 10 LCDR3 12 LCDR3 36 V _H 14 V _H 56 V _L 16 V _L 58 scFv 110 scFv 126 ALG.APV-199 (scFv-Fc-scFv format) Binding domain to 4-1BB Binding domain to 5T4 Component SEQ ID NO: Component SEQ ID NO: HCDR1 2 HCDR1 60 HCDR2 4 HCDR2 62 HCDR3 6 HCDR3 34 LCDR1 8 LCDR1 54 LCDR2 10 LCDR2 10 LCDR3 12 LCDR3 36 V _H 14 V _H 64 V _L 16 V _L 58 scFv 110 scFv 128 ALG.APV-006 (scFv-Fc-scFv format) Binding domain to 4-1BB Binding domain to 5T4 Component SEQ ID NO: Component SEQ ID NO: HCDR1 24 HCDR1 thirty HCDR2 4 HCDR2 32 HCDR3 6 HCDR3 34 LCDR1 8 LCDR1 8 LCDR2 10 LCDR2 10 LCDR3 12 LCDR3 36 V _H 28 V _H 38 V _L 16 V _L 68 scFv 116 scFv 130 ALG.APV-010 (scFv-Fc-scFv format) Binding domain to 4-1BB Binding domain to 5T4 Component SEQ ID NO: Component SEQ ID NO: HCDR1 24 HCDR1 thirty HCDR2 4 HCDR2 32 HCDR3 6 HCDR3 34 LCDR1 8 LCDR1 8 LCDR2 10 LCDR2 10 LCDR3 12 LCDR3 36 V _H 28 V _H 38 V _L 16 V _L 68 scFv 116 scFv 170 ALG.APV-014 (scFv-Fc-scFv format) Binding domain to 4-1BB Binding domain to 5T4 Component SEQ ID NO: Component SEQ ID NO: HCDR1 24 HCDR1 thirty HCDR2 4 HCDR2 32 HCDR3 6 HCDR3 34 LCDR1 8 LCDR1 8 LCDR2 10 LCDR2 10 LCDR3 12 LCDR3 36 V _H 28 V _H 46 V _L 16 V _L 70 scFv 116 scFv 132 ALG.APV-018 (scFv-Fc-scFv format) Binding domain to 4-1BB Binding domain to 5T4 Component SEQ ID NO: Component SEQ ID NO: HCDR1 24 HCDR1 thirty HCDR2 4 HCDR2 32 HCDR3 6 HCDR3 34 LCDR1 8 LCDR1 8 LCDR2 10 LCDR2 10 LCDR3 12 LCDR3 36 V _H 28 V _H 46 V _L 16 V _L 70 scFv 116 scFv 134

[00223] In some embodiments, the 5T4 x 4-1BB molecules described herein may comprise at least about 90%, at least about 91%, at least about 92% amino acid sequence of at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 172, 174 and 176. In some embodiments, the 5T4 x 4-1BB molecules described herein may comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 172, 174 and 176. Exemplary amino acid and nucleotide sequences of the 5T4 x 4-1BB molecules described herein are presented in Tables 10 and 11. In each of Tables 10 and 11 immunoglobulin Fc domains (hinge - CH1 - CH2) are indicated in underlined text, and the binding domain linker sequences are indicated in bold.

Table 10. Illustrative DNA sequences of the 5T4 x 4-1BB molecule

Design DNA sequence SEQ ID NO: DNA sequences ALG.APV-178 atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctgttggagagcgggggaggcttggtacagcctggggggtccctgcgcctctcctgtgcagccagcggattcaccttttctcacggttctatgtactgggtccgccaggctccagggaaggggctggagtgggtctcatctatttcttctggttctggttctacatactatgcagactccgtgaagggccggttcaccatctcccatgacaattccaagaacacgctgtatctgcaaatgaacagcctgcgtgccgaggacacggctgtatattattgtgcgcgctcttcttactacggttcttactactctattgactattggggccagggaaccctggtcaccgtctcctcaggtggaggtggctccgggggtggaggttccggaggaggcggatcaggtggaggcggaagcgacatccagatgacccagtctccatcctccctgagcgcatctgtaggagaccgcgtcaccatcacttgccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtttgcaaagtggggtcccatcacgtttcagtggcagtggaagcgggacagatttcactctcaccatcagcagtctacaacctgaagattttgcaacttattactgtcaacagtactacgacaacctgcccacttttggccaggggaccaagctggagatcaaatcgagtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggaatacaagtgcgcggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggttccggaggtggcggttcgggaggtggcgggtcaggaggtgggggatccccttcagaagtgcagctgctggagtccggaggaggactggtgcagcctggcggaagcctgaggctgagctgcgctgcctccggcttcacattcagcagctatgctatgagctgggtgaggcaagcccctggaaagggcctggagtgggtgtccgctatctccggcagcggcggaagcacctactacgctgactccgtcaagggcaggttcaccatcagccgggacaacagcaagaacaccctgtacctgcagatgaatagcctcagggctgaagacaccgctgtgtactactgcgccaggtactatggcggctactactccgcctggatggactactggggacagggcacactggtgaccgtgtccagcggcggaggcggctccggaggcggtggctccggaggaggcggaagcggaggaggaggctccgatattcagatgacacagtcccctagctccctgtccgccagcgtgggagatcgggtgaccatcacctgcagggccagccagtccatctccagctatttaaactggtaccagcagaagcctggaaaggctcccaagctgctgatctacgccgcttccagcctccagagcggcgtgcctagcaggttctccggctccggaagcggaacagacttcaccctgaccatcagctccctgcagcccgaggactccgctacctactactgccagcagacctatggctacctgcacaccttcggccagggcacaaagctggagatcaagcgc 135 ALG.APV-179 atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctgttggagagcgggggaggcttggtacagcctggggggtccctgcgcctctcctgtgcagccagcggattcaccttttctcacggttctatgtactgggtccgccaggctccagggaaggggctggagtgggtctcatctatttcttctggttctggttctacatactatgcagactccgtgaagggccggttcaccatctcccatgacaattccaagaacacgctgtatctgcaaatgaacagcctgcgtgccgaggacacggctgtatattattgtgcgcgctcttcttactacggttcttactactctattgactattggggccagggaaccctggtcaccgtctcctcaggtggaggtggctccgggggtggaggttccggaggaggcggatcaggtggaggcggaagcgacatccagatgacccagtctccatcctccctgagcgcatctgtaggagaccgcgtcaccatcacttgccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtttgcaaagtggggtcccatcacgtttcagtggcagtggaagcgggacagatttcactctcaccatcagcagtctacaacctgaagattttgcaacttattactgtcaacagtactacgacaacctgcccacttttggccaggggaccaagctggagatcaaatcgagtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggaatacaagtgcgcggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggttccggaggtggcggttcgggaggtggcgggtcaggaggtgggggatccccttcagaagtgcagctgctggagtccggaggaggactggtgcagcctggcggaagcctgaggctgagctgcgctgcctccggcttcacattcagcagctatgctatgagctgggtgaggcaagcccctggaaagggcctggagtgggtgtccgctatctccggcagcggcggaagcacctactacgctgactccgtcaagggcaggttcaccatcagccgggacaacagcaagaacaccctgtacctgcagatgaatagcctcagggctgaagacaccgctgtgtactactgcgccaggtactatggcggctactactccgcctggatggactactggggacagggcacactggtgaccgtgtccagcggcggaggcggctccggaggcggtggctccggaggaggcggaagcggaggaggaggctccgatattcagatgacacagtcccctagctccctgtccgccagcgtgggagatcgggtgaccatcacctgcagggccagccagtccatctccagcttcttaaactggtaccagcagaagcctggaaaggctcccaagctgctgatctacgccgcttccagcctccagagcggcgtgcctagcaggttctccggctccggaagcggaacagacttcaccctgaccatcagctccctgcagcccgaggactccgctacctactactgccagcagacctatggctacctgcacaccttcggccagggcacaaagctggagatcaagcgc 137 ALG.APV-187 atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctgttggagagcgggggaggcttggtacagcctggggggtccctgcgcctctcctgtgcagccagcggattcaccttttctcacggttctatgtactgggtccgccaggctccagggaaggggctggagtgggtctcatctatttcttctggttctggttctacatactatgcagactccgtgaagggccggttcaccatctcccatgacaattccaagaacacgctgtatctgcaaatgaacagcctgcgtgccgaggacacggctgtatattattgtgcgcgctcttcttactacggttcttactactctattgactattggggccagggaaccctggtcaccgtctcctcaggtggaggtggctccgggggtggaggttccggaggaggcggatcaggtggaggcggaagcgacatccagatgacccagtctccatcctccctgagcgcatctgtaggagaccgcgtcaccatcacttgccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtttgcaaagtggggtcccatcacgtttcagtggcagtggaagcgggacagatttcactctcaccatcagcagtctacaacctgaagattttgcaacttattactgtcaacagtactacgacaacctgcccacttttggccaggggaccaagctggagatcaaatcgagtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggaatacaagtgcgcggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggttccggaggtggcggttcgggaggtggcgggtcaggaggtgggggatccccttcagaagtgcagctgctggagtccggaggaggactggtgcagcctggcggaagcctgaggctgagctgcgctgcctccggcttcacattcagcagctatgctatgagctgggtgaggcaagcccctggaaagtgcctggagtgggtgtccgctatctccggcagcggcggaagcacctactacgctgactccgtcaagggcaggttcaccatcagccgggacaacagcaagaacaccctgtacctgcagatgaatagcctcagggctgaagacaccgctgtgtactactgcgccaggtactatggcggctactactccgcctggatggactactggggacagggcacactggtgaccgtgtccagcggcggaggcggctccggaggcggtggctccggaggaggcggaagcggaggaggaggctccgatattcagatgacacagtcccctagctccctgtccgccagcgtgggagatcgggtgaccatcacctgcagggccagccagtccatctccagcttcttaaactggtaccagcagaagcctggaaaggctcccaagctgctgatctacgccgcttccagcctccagagcggcgtgcctagcaggttctccggctccggaagcggaacagacttcaccctgaccatcagctccctgcagcccgaggactccgctacctactactgccagcagacctatggctacctgcacaccttcggctgcggcacaaagctggagatcaagcgc 139 ALG.APV-191 atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctgttggagagcgggggaggcttggtacagcctggggggtccctgcgcctctcctgtgcagccagcggattcacctttgattacggttctatgtactgggtccgccaggctccagggaaggggctggagtgggtctcatctatttcttctggttctggttctacatactatgcagactccgtgaagggccggttcaccatctcccacgacaattccaagaacacgctgtatctgcaaatgaacagcctgcgtgccgaggacacggctgtatattattgtgcgcgctcttcttactacggttcttactactctattgactattggggccagggaaccctggtcaccgtctcctcaggtggaggtggctccgggggtggaggttccggaggaggcggatcaggtggaggcggaagcgacatccagatgacccagtctccatcctccctgagcgcatctgtaggagaccgcgtcaccatcacttgccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtttgcacagtggggtcccatcacgtttcagtggcagtggaagcgggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttattactgtcaacagtactacgacaacctgcccacttttggccaggggaccaagctggagatcaaatcctcggagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggaatacaagtgcgcggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggttccggaggtggcggttcgggaggtggcgggtcaggaggtgggggatccccttcagaagtgcagctgctggagtccggaggaggactggtgcagcctggcggaagcctgaggctgagctgcgctgcctccggcttcacattcagcagctatgctatgagctgggtgaggcaagcccctggaaagtgcctggagtgggtgtccgctatctccggcagcggcggaagcacctactacgctgactccgtcaagggcaggttcaccatcagccgggacaacagcaagaacaccctgtacctgcagatgaatagcctcagggctgaagacaccgctgtgtactactgcgccaggtactatggcggctactactccgcctggatggactactggggacagggcacactggtgaccgtgtccagcggcggaggcggctccggaggcggtggctccggaggaggcggaagcggaggaggaggctccgatattcagatgacacagtcccctagctccctgtccgccagcgtgggagatcgggtgaccatcacctgcagggccagccagtccatctccagcttcttaaactggtaccagcagaagcctggaaaggctcccaagctgctgatctacgccgcttccagcctccagagcggcgtgcctagcaggttctccggctccggaagcggaacagacttcaccctgaccatcagctccctgcagcccgaggacttcgctacctactactgccagcagacctatggctacctgcacaccttcggctgcggcacaaagctggagatcaagagc 141 ALG.APV-196 atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctgttggagagcgggggaggcttggtacagcctggggggtccctgcgcctctcctgtgcagccagcggattcaccttttcttacggttctatgtactgggtccgccaggctccagggaaggggctggagtgggtctcatctatttcttctggttctggttctacatactatgcagactccgtgaagggccggttcaccatctcccatgacaattccaagaacacgctgtatctgcaaatgaacagcctgcgtgccgaggacacggctgtatattattgtgcgcgctcttcttactacggttcttactactctattgactattggggccagggaaccctggtcaccgtctcctcaggtggaggtggctccgggggtggaggttccggaggaggcggatcaggtggaggcggaagcgacatccagatgacccagtctccatcctccctgagcgcatctgtaggagaccgcgtcaccatcacttgccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtttgcaaagtggggtcccatcacgtttcagtggcagtggaagcgggacagatttcactctcaccatcagcagtctacaacctgaagattttgcaacttattactgtcaacagtactacgacaacctgcccacttttggccaggggaccaagctggagatcaaatcctcggagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggaatacaagtgcgcggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggttccggaggtggcggttcgggaggtggcgggtcaggaggtgggggatccccttcagaagtgcagctgctggagtccggaggaggactggtgcagcctggcggaagcctgaggctgagctgcgctgcctccggcttcgacttcgagagctatgctatgagctgggtgaggcaagcccctggaaagtgcctggagtgggtgtccgctatctccggcagcggcggaagcacctactacgctgactccgtcaagggcaggttcaccatcagccgggacaacagcaagaacaccctgtacctgcagatgaatagcctcagggctgaagacaccgctgtgtactactgcgccaggtactatggcggctactactccgcctggatggactactggggacagggcacactggtgaccgtgtccagcggcggaggcggctccggaggcggtggctccggaggaggcggaagcggaggaggaggctccgatattcagatgacacagtcccctagctccctgtccgccagcgtgggagatcgggtgaccatcacctgcagggccagccagtccatcaggagcgccctgaactggtaccagcagaagcctggaaaggctcccaagctgctgatctacgccgcttccagcctccagagcggcgtgcctagcaggttctccggctccggaagcggaacagacttcaccctgaccatcagctccctgcagcccgaggacttcgctacctactactgccagcagacctatggctacctgcacaccttcggctgcggcacaaagctggagatcaagcgc 143 ALG.APV-198 atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctgttggagagcgggggaggcttggtacagcctggggggtccctgcgcctctcctgtgcagccagcggattcaccttttctcacggttctatgtactgggtccgccaggctccagggaaggggctggagtgggtctcatctatttcttctggttctggttctacatactatgcagactccgtgaagggccggttcaccatctcccatgacaattccaagaacacgctgtatctgcaaatgaacagcctgcgtgccgaggacacggctgtatattattgtgcgcgctcttcttactacggttcttactactctattgactattggggccagggaaccctggtcaccgtctcctcaggtggaggtggctccgggggtggaggttccggaggaggcggatcaggtggaggcggaagcgacatccagatgacccagtctccatcctccctgagcgcatctgtaggagaccgcgtcaccatcacttgccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtttgcaaagtggggtcccatcacgtttcagtggcagtggaagcgggacagatttcactctcaccatcagcagtctacaacctgaagattttgcaacttattactgtcaacagtactacgacaacctgcccacttttggccaggggaccaagctggagatcaaatcgagtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggaatacaagtgcgcggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggttccggaggtggcggttcgggaggtggcgggtcaggaggtgggggatccccttcagaagtgcagctgctggagtccggaggaggactggtgcagcctggcggaagcctgaggctgagctgcgctgcctccggcttcgacttcgagagctatgctatgagctgggtgaggcaagcccctggaaagtgcctggagtgggtgtccgctatctccggcagcggcggaagcacctactacgctgactccgtcaagggcaggttcaccatcagccgggacaacagcaagaacaccctgtacctgcagatgaatagcctcagggctgaagacaccgctgtgtactactgcgccaggtactatggcggctactactccgcctggatggactactggggacagggcacactggtgaccgtgtccagcggcggaggcggctccggaggcggtggctccggaggaggcggaagcggaggaggaggctccgatattcagatgacacagtcccctagctccctgtccgccagcgtgggagatcgggtgaccatcacctgcagggccagccagtccatcaggagcgccctgaactggtaccagcagaagcctggaaaggctcccaagctgctgatctacgccgcttccagcctccagagcggcgtgcctagcaggttctccggctccggaagcggaacagacttcaccctgaccatcagctccctgcagcccgaggacttcgctacctactactgccagcagacctatggctacctgcacaccttcggctgcggcacaaagctggagatcaagcgc 145 ALG.APV-199 atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctgttggagagcgggggaggcttggtacagcctggggggtccctgcgcctctcctgtgcagccagcggattcaccttttctcacggttctatgtactgggtccgccaggctccagggaaggggctggagtgggtctcatctatttcttctggttctggttctacatactatgcagactccgtgaagggccggttcaccatctcccatgacaattccaagaacacgctgtatctgcaaatgaacagcctgcgtgccgaggacacggctgtatattattgtgcgcgctcttcttactacggttcttactactctattgactattggggccagggaaccctggtcaccgtctcctcaggtggaggtggctccgggggtggaggttccggaggaggcggatcaggtggaggcggaagcgacatccagatgacccagtctccatcctccctgagcgcatctgtaggagaccgcgtcaccatcacttgccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtttgcaaagtggggtcccatcacgtttcagtggcagtggaagcgggacagatttcactctcaccatcagcagtctacaacctgaagattttgcaacttattactgtcaacagtactacgacaacctgcccacttttggccaggggaccaagctggagatcaaatcgagtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggaatacaagtgcgcggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggttccggaggtggcggttcgggaggtggcgggtcaggaggtgggggatccccttcagaagtgcagctgctggagtccggaggaggactggtgcagcctggcggaagcctgaggctgagctgcgctgcctccggcttcgacttcgacagctatgctatgagctgggtgaggcaagcccctggaaagtgcctggagtgggtgtccgctatctccggcaggggcggaagcacctactacgctgactccgtcaagggcaggttcaccatcagccgggacaacagcaagaacaccctgtacctgcagatgaatagcctcagggctgaagacaccgctgtgtactactgcgccaggtactatggcggctactactccgcctggatggactactggggacagggcacactggtgaccgtgtccagcggcggaggcggctccggaggcggtggctccggaggaggcggaagcggaggaggaggctccgatattcagatgacacagtcccctagctccctgtccgccagcgtgggagatcgggtgaccatcacctgcagggccagccagtccatcaggagcgccctgaactggtaccagcagaagcctggaaaggctcccaagctgctgatctacgccgcttccagcctccagagcggcgtgcctagcaggttctccggctccggaagcggaacagacttcaccctgaccatcagctccctgcagcccgaggacttcgctacctactactgccagcagacctatggctacctgcacaccttcggctgcggcacaaagctggagatcaagcgc 147 ALG.APV-208 atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctgttggagagcgggggaggcttggtacagcctggggggtccctgcgcctctcctgtgcagccagcggattcaccttttctcacggttctatgtactgggtccgccaggctccagggaaggggctggagtgggtctcatctatttcttctggttctggttctacatactatgcagactccgtgaagggccggttcaccatctcccatgacaattccaagaacacgctgtatctgcaaatgaacagcctgcgtgccgaggacacggctgtatattattgtgcgcgctcttcttactacggttcttactactctattgactattggggccagggaaccctggtcaccgtctcctcaggtggaggtggctccgggggtggaggttccggaggaggcggatcaggtggaggcggaagcgacatccagatgacccagtctccatcctccctgagcgcatctgtaggagaccgcgtcaccatcacttgccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtttgcaaagtggggtcccatcacgtttcagtggcagtggaagcgggacagatttcactctcaccatcagcagtctacaacctgaagattttgcaacttattactgtcaacagtactacgacaacctgcccacttttggccaggggaccaagctggagatcaaagagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggaatacaagtgcgcggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggttccggaggtggcggttcgggaggtggcgggtcaggaggtgggggatccccttcagaagtgcagctgctggagtccggaggaggactggtgcagcctggcggaagcctgaggctgagctgcgctgcctccggcttcacattcagcagctatgctatgagctgggtgaggcaagcccctggaaagggcctggagtgggtgtccgctatctccggcagcggcggaagcacctactacgctgactccgtcaagggcaggttcaccatcagccgggacaacagcaagaacaccctgtacctgcagatgaatagcctcagggctgaagacaccgctgtgtactactgcgccaggtactatggcggctactactccgcctggatggactactggggacagggcacactggtgaccgtgtccagcggcggaggcggctccggaggcggtggctccggaggaggcggaagcggaggaggaggctccgatattcagatgacacagtcccctagctccctgtccgccagcgtgggagatcgggtgaccatcacctgcagggccagccagtccatctccagctatttaaactggtaccagcagaagcctggaaaggctcccaagctgctgatctacgccgcttccagcctccagagcggcgtgcctagcaggttctccggctccggaagcggaacagacttcaccctgaccatcagctccctgcagcccgaggactccgctacctactactgccagcagacctatggctacctgcacaccttcggccagggcacaaagctggagatcaagcgc 175 ALG.APV-209
ALG.APV-222 ATGGAAGCACCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGTGAGGTGCAGCTGTTGGAGAGCGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAGCCAGCGGATTCACCTTTTCTCACGGTTCTATGTACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCTATTTCTTCTGGTTCTGGTTCTACATACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCATGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCTCTTCTTACTACGGTTCTTACTACTCTATTGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGGAGGTGGCTCCGGGGGTGGAGGTTCCGGAGGAGGCGGATCAGGTGGAGGCGGAAGCGACATCCAGATGACCCAGTCTCCATCCTCCCTGAGCGCATCTGTAGGAGACCGCGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTACAACCTGAAGATTTTGCAACTTATTACTGTCAACAGTACTACGACAACCTGCCCACTTTTGGCCAGGGGACCAAGCTGGAGATCAAAGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGCGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAATACAAGTGCGCGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTTCCGGAGGTGGCGGTTCGGGAGGTGGCGGGTCAGGAGGTGGGGGATCCCCTTCAGAAGTGCAGCTGCTGGAGTCCGGAGGAGGACTGGTGCAGCCTGGCGGAAGCCTGAGGCTGAGCTGCGCTGCCTCCGGCTTCACATTCAGCAGCTATGCTATGAGCTGGGTGAGGCAAGCCCCTGGAAAGGGCCTGGAGTGGGTGTCCGCTATCTCCGGCAGCGGCGGAAGCACCTACTACGCTGACTCCGTCAAGGGCAGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAATAGCCTCAGGGCTGAAGACACCGCTGTGTACTACTGCGCCAGGTACTATGGCGGCTACTACTCCGCCTGGATGGACTACTGGGGACAGGGCACACTGGTGACCGTGTCCAGCGGCGGAGGCGGCTCCGGAGGCGGTGGCTCCGGAGGAGGCGGAAGCGGAGGAGGAGGCTCCGATATTCAGATGACACAGTCCCCTAGCTCCCTGTCCGCCAGCGTGGGAGATCGGGTGACCATCACCTGCAGGGCCAGCCAGTCCATCTCCAGCTTCTTAAACTGGTACCAGCAGAAGCCTGGAAAGGCTCCCAAGCTGCTGATCTACGCCGCTTCCAGCCTCCAGAGCGGCGTGCCTAGCAGGTTCTCCGGCTCCGGAAGCGGAACAGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTCCGCTACCTACTACTGCCAGCAGACCTATGGCTACCTGCACACCTTCGGCCAGGGCACAAAGCTGGAGATCAAGCGC 171 ALG.APV-210
ALG.APV-223 ATGGAAGCACCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGTGAGGTGCAGCTGTTGGAGAGCGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAGCCAGCGGATTCACCTTTTCTCACGGTTCTATGTACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCTATTTCTTCTGGTTCTGGTTCTACATACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCATGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCTCTTCTTACTACGGTTCTTACTACTCTATTGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGGAGGTGGCTCCGGGGGTGGAGGTTCCGGAGGAGGCGGATCAGGTGGAGGCGGAAGCGACATCCAGATGACCCAGTCTCCATCCTCCCTGAGCGCATCTGTAGGAGACCGCGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTACAACCTGAAGATTTTGCAACTTATTACTGTCAACAGTACTACGACAACCTGCCCACTTTTGGCCAGGGGACCAAGCTGGAGATCAAAGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGCGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAATACAAGTGCGCGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTTCCGGAGGTGGCGGTTCGGGAGGTGGCGGGTCAGGAGGTGGGGGATCCCCTTCAGAAGTGCAGCTGCTGGAGTCCGGAGGAGGACTGGTGCAGCCTGGCGGAAGCCTGAGGCTGAGCTGCGCTGCCTCCGGCTTCACATTCAGCAGCTATGCTATGAGCTGGGTGAGGCAAGCCCCTGGAAAGTGCCTGGAGTGGGTGTCCGCTATCTCCGGCAGCGGCGGAAGCACCTACTACGCTGACTCCGTCAAGGGCAGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAATAGCCTCAGGGCTGAAGACACCGCTGTGTACTACTGCGCCAGGTACTATGGCGGCTACTACTCCGCCTGGATGGACTACTGGGGACAGGGCACACTGGTGACCGTGTCCAGCGGCGGAGGCGGCTCCGGAGGCGGTGGCTCCGGAGGAGGCGGAAGCGGAGGAGGAGGCTCCGATATTCAGATGACACAGTCCCCTAGCTCCCTGTCCGCCAGCGTGGGAGATCGGGTGACCATCACCTGCAGGGCCAGCCAGTCCATCTCCAGCTTCTTAAACTGGTACCAGCAGAAGCCTGGAAAGGCTCCCAAGCTGCTGATCTACGCCGCTTCCAGCCTCCAGAGCGGCGTGCCTAGCAGGTTCTCCGGCTCCGGAAGCGGAACAGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTCCGCTACCTACTACTGCCAGCAGACCTATGGCTACCTGCACACCTTCGGCTGCGGCACAAAGCTGGAGATCAAGCGC 173 ALG.APV-006 atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctgttggagagcgggggaggcttggtacagcctggggggtccctgcgcctctcctgtgcagccagcggattcaccttttcttacggttctatgtactgggtccgccaggctccagggaaggggctggagtgggtctcatctatttcttctggttctggttctacatactatgcagactccgtgaagggccggttcaccatctcccgtgacaattccaagaacacgctgtatctgcaaatgaacagcctgcgtgccgaggacacggctgtatattattgtgcgcgctcttcttactacggttcttactactctattgactattggggccagggaaccctggtcaccgtctcctcaggcggcggcggcagcggcggcggcggcagcggcggcggaggctccggcggcggcggcagcgacatccagatgacccagtctccatcctccctgagcgcatctgtaggagaccgcgtcaccatcacttgccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtttgcaaagtggggtcccatcacgtttcagtggcagtggaagcgggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttattactgtcaacagtactacgacaacctgcccacttttggccaggggaccaagctggagatcaaatcctcgagtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggaatacaagtgcgcggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggttccggaggtggcggttcgggaggtggcgggtcaggaggtgggggatccccttcagaggtgcagctgttggagagcgggggaggcttggtacagcctggggggtccctgcgcctctcctgtgcagccagcggattcacctttagcagctatgccatgagctgggtccgccaggctccagggaaggggctggagtgggtctcagctattagtggtagtggtggtagcacatactatgcagactccgtgaagggccggttcaccatctcccgtgacaattccaagaacacgctgtatctgcaaatgaacagcctgcgtgccgaggacacggctgtatattattgtgcgcgctactacggtggttactactctgcttggatggactattggggccagggaaccctggtcaccgtctcctcaggcggtggaggcagcggtgggggtgggtctggaggcggtggcagtggcggcggaggctctgacatccagatgacccagtctccatcctccctgagcgcatctgtaggagaccgcgtcaccatcacttgccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtttgcaaagtggggtcccatcacgtttcagtggcagtggaagcgggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttattactgtcaacagacttacggttacctgcacacttttggccaggggaccaagctggagatcaaatca 149 ALG.APV-010 atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctgttggagagcgggggaggcttggtacagcctggggggtccctgcgcctctcctgtgcagccagcggattcaccttttcttacggttctatgtactgggtccgccaggctccagggaaggggctggagtgggtctcatctatttcttctggttctggttctacatactatgcagactccgtgaagggccggttcaccatctcccgtgacaattccaagaacacgctgtatctgcaaatgaacagcctgcgtgccgaggacacggctgtatattattgtgcgcgctcttcttactacggttcttactactctattgactattggggccagggaaccctggtcaccgtctcctcaggcggcggcggcagcggcggcggcggcagcggcggcggaggctccggcggcggcggcagcgacatccagatgacccagtctccatcctccctgagcgcatctgtaggagaccgcgtcaccatcacttgccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtttgcaaagtggggtcccatcacgtttcagtggcagtggaagcgggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttattactgtcaacagtactacgacaacctgcccacttttggccaggggaccaagctggagatcaaatcctcgagtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggaatacaagtgcgcggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggttccggaggtggcggttcgggaggtggcgggtcaggaggtgggggatccccttcagacatccagatgacccagtctccatcctccctgagcgcatctgtaggagaccgcgtcaccatcacttgccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtttgcaaagtggggtcccatcacgtttcagtggcagtggaagcgggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttattactgtcaacagacttacggttacctgcacacttttggccaggggaccaagctggagatcaaaggcggtggaggcagcggtgggggtgggtctggaggcggtggcagtggcggcggaggctctgaggtgcagctgttggagagcgggggaggcttggtacagcctggggggtccctgcgcctctcctgtgcagccagcggattcacctttagcagctatgccatgagctgggtccgccaggctccagggaaggggctggagtgggtctcagctattagtggtagtggtggtagcacatactatgcagactccgtgaagggccggttcaccatctcccgtgacaattccaagaacacgctgtatctgcaaatgaacagcctgcgtgccgaggacacggctgtatattattgtgcgcgctactacggtggttactactctgcttggatggactattggggccagggaaccctggtcaccgtctcctca 151 ALG.APV-014 atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctgttggagagcgggggaggcttggtacagcctggggggtccctgcgcctctcctgtgcagccagcggattcaccttttcttacggttctatgtactgggtccgccaggctccagggaaggggctggagtgggtctcatctatttcttctggttctggttctacatactatgcagactccgtgaagggccggttcaccatctcccgtgacaattccaagaacacgctgtatctgcaaatgaacagcctgcgtgccgaggacacggctgtatattattgtgcgcgctcttcttactacggttcttactactctattgactattggggccagggaaccctggtcaccgtctcctcaggcggcggcggcagcggcggcggcggcagcggcggcggaggctccggcggcggcggcagcgacatccagatgacccagtctccatcctccctgagcgcatctgtaggagaccgcgtcaccatcacttgccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtttgcaaagtggggtcccatcacgtttcagtggcagtggaagcgggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttattactgtcaacagtactacgacaacctgcccacttttggccaggggaccaagctggagatcaaatcctcgagtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggaatacaagtgcgcggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggttccggaggtggcggttcgggaggtggcgggtcaggaggtgggggatccccttcagaggtgcagctgttggagagcgggggaggcttggtacagcctggggggtccctgcgcctctcctgtgcagccagcggattcacctttagcagctatgccatgagctgggtccgccaggctccagggaagtgcctggagtgggtctcagctattagtggtagtggtggtagcacatactatgcagactccgtgaagggccggttcaccatctcccgtgacaattccaagaacacgctgtatctgcaaatgaacagcctgcgtgccgaggacacggctgtatattattgtgcgcgctactacggtggttactactctgcttggatggactattggggccagggaaccctggtcaccgtctcctcaggcggtggaggcagcggtgggggtgggtctggaggcggtggcagtggcggcggaggctctgacatccagatgacccagtctccatcctccctgagcgcatctgtaggagaccgcgtcaccatcacttgccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtttgcaaagtggggtcccatcacgtttcagtggcagtggaagcgggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttattactgtcaacagacttacggttacctgcacacttttggctgcgggaccaagctggagatcaaatca 153 ALG.APV-018 atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctgttggagagcgggggaggcttggtacagcctggggggtccctgcgcctctcctgtgcagccagcggattcaccttttcttacggttctatgtactgggtccgccaggctccagggaaggggctggagtgggtctcatctatttcttctggttctggttctacatactatgcagactccgtgaagggccggttcaccatctcccgtgacaattccaagaacacgctgtatctgcaaatgaacagcctgcgtgccgaggacacggctgtatattattgtgcgcgctcttcttactacggttcttactactctattgactattggggccagggaaccctggtcaccgtctcctcaggcggcggcggcagcggcggcggcggcagcggcggcggaggctccggcggcggcggcagcgacatccagatgacccagtctccatcctccctgagcgcatctgtaggagaccgcgtcaccatcacttgccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtttgcaaagtggggtcccatcacgtttcagtggcagtggaagcgggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttattactgtcaacagtactacgacaacctgcccacttttggccaggggaccaagctggagatcaaatcctcgagtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggaatacaagtgcgcggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggttccggaggtggcggttcgggaggtggcgggtcaggaggtgggggatccccttcagacatccagatgacccagtctccatcctccctgagcgcatctgtaggagaccgcgtcaccatcacttgccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtttgcaaagtggggtcccatcacgtttcagtggcagtggaagcgggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttattactgtcaacagacttacggttacctgcacacttttggctgcgggaccaagctggagatcaaaggcggtggaggcagcggtgggggtgggtctggaggcggtggcagtggcggcggaggctctgaggtgcagctgttggagagcgggggaggcttggtacagcctggggggtccctgcgcctctcctgtgcagccagcggattcacctttagcagctatgccatgagctgggtccgccaggctccagggaagtgcctggagtgggtctcagctattagtggtagtggtggtagcacatactatgcagactccgtgaagggccggttcaccatctcccgtgacaattccaagaacacgctgtatctgcaaatgaacagcctgcgtgccgaggacacggctgtatattattgtgcgcgctactacggtggttactactctgcttggatggactattggggccagggaaccctggtcaccgtctcctca 155

Table 11. Exemplary amino acid sequences of the 5T4 x 4-1BB molecule

Design Amino acid sequence SEQ ID NO: amino acid sequence ALG.APV-178 MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSHGSMYWVRQAPGKGLEWVSSISSGSGSTYYADSVKGRFTISHDNSKNTLYLQMNSLRAEDTAVYYCARSSYYGSYYSIDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGK APKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYDNLPTFGQGTKLEIKSSEPKSSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSGGGGSPSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAP KLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDSATYYCQQTYGYLHTFGQGTKLEIKR 136 ALG.APV-179 MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSHGSMYWVRQAPGKGLEWVSSISSGSGSTYYADSVKGRFTISHDNSKNTLYLQMNSLRAEDTAVYYCARSSYYGSYYSIDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGK APKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYDNLPTFGQGTKLEIKSSEPKSSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSGGGGSPSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSFLNWYQQKPGKAP KLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDSATYYCQQTYGYLHTFGQGTKLEIKR 138 ALG.APV-187 MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSHGSMYWVRQAPGKGLEWVSSISSGSGSTYYADSVKGRFTISHDNSKNTLYLQMNSLRAEDTAVYYCARSSYYGSYYSIDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGK APKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYDNLPTFGQGTKLEIKSSEPKSSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSGGGGSPSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKCLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSFLNWYQQKPGKAP KLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDSATYYCQQTYGYLHTFGCGTKLEIKR 140 ALG.APV-191 MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFDYGSMYWVRQAPGKGLEWVSSISSGSGSTYYADSVKGRFTISHDNSKNTLYLQMNSLRAEDTAVYYCARSSYYGSYYSIDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPG KAPKLLIYAASSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYDNLPTFGQGTKLEIKSSEPKSSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSGGGGSPSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKCLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSFLNWYQQKPGKAP KLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYGYLHTFGCGTKLEIKS 142 ALG.APV-196 MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSYGSMYWVRQAPGKGLEWVSSISSGSGSTYYADSVKGRFTISHDNSKNTLYLQMNSLRAEDTAVYYCARSSYYGSYYSIDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGK APKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYDNLPTFGQGTKLEIKSSEPKSSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSGGGGSPSEVQLLESGGGLVQPGGSLRLSCAASGFDFESYAMSWVRQAPGKCLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSIRSALNWYQQKPGKAP KLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYGYLHTFGCGTKLEIKR 144 ALG.APV-198 MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSHGSMYWVRQAPGKGLEWVSSISSGSGSTYYADSVKGRFTISHDNSKNTLYLQMNSLRAEDTAVYYCARSSYYGSYYSIDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGK APKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYDNLPTFGQGTKLEIKSSEPKSSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSGGGGSPSEVQLLESGGGLVQPGGSLRLSCAASGFDFESYAMSWVRQAPGKCLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSIRSALNWYQQKPGKAP KLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYGYLHTFGCGTKLEIKR 146 ALG.APV-199 MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSHGSMYWVRQAPGKGLEWVSSISSGSGSTYYADSVKGRFTISHDNSKNTLYLQMNSLRAEDTAVYYCARSSYYGSYYSIDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGK APKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYDNLPTFGQGTKLEIKSSEPKSSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSGGGGSPSEVQLLESGGGLVQPGGSLRLSCAASGFDFDSYAMSWVRQAPGKCLEWVSAISGRGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSIRSALNWYQQKPGKAP KLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYGYLHTFGCGTKLEIKR 148 ALG.APV-208 MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSHGSMYWVRQAPGKGLEWVSSISSGSGSTYYADSVKGRFTISHDNSKNTLYLQMNSLRAEDTAVYYCARSSYYGSYYSIDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGK APKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYDNLPTFGQGTKLEIKEPKSSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSGGGGSPSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAP KLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDSATYYCQQTYGYLHTFGQGTKLEIKR 176 ALG.APV-209
ALG.APV-222 MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSHGSMYWVRQAPGKGLEWVSSISSGSGSTYYADSVKGRFTISHDNSKNTLYLQMNSLRAEDTAVYYCARSSYYGSYYSIDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGK APKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYDNLPTFGQGTKLEIKEPKSSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSGGGGSPSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSFLNWYQQKPGKAP KLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDSATYYCQQTYGYLHTFGQGTKLEIKR 172 ALG.APV-210
ALG.APV-223 MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSHGSMYWVRQAPGKGLEWVSSISSGSGSTYYADSVKGRFTISHDNSKNTLYLQMNSLRAEDTAVYYCARSSYYGSYYSIDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGK APKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYDNLPTFGQGTKLEIKEPKSSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSGGGGSPSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKCLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSFLNWYQQKPGKAP KLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDSATYYCQQTYGYLHTFGCGTKLEIKR 174 ALG.APV-006 MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSYGSMYWVRQAPGKGLEWVSSISSGSGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSSYYGSYYSIDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGK APKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYDNLPTFGQGTKLEIKSSSEPKSSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSGGGGSPSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAP KLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYGYLHTFGQGTKLEIKS 150 ALG.APV-010 MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSYGSMYWVRQAPGKGLEWVSSISSGSGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSSYYGSYYSIDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGK APKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYDNLPTFGQGTKLEIKSSSEPKSSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSGGGGSPSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYGYLHTFGQGTKLEIKGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGS GGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSS 152 ALG.APV-014 MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSYGSMYWVRQAPGKGLEWVSSISSGSGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSSYYGSYYSIDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGK APKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYDNLPTFGQGTKLEIKSSSEPKSSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSGGGGSPSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKCLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAP KLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYGYLHTFGCGTKLEIKS 154 ALG.APV-018 MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSYGSMYWVRQAPGKGLEWVSSISSGSGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSSYYGSYYSIDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGK APKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYDNLPTFGQGTKLEIKSSSEPKSSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSGGGGSPSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYGYLHTFGCGTKLEIKGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFFSSYAMSWVRQAPGKCLEWVSAISGSGG STYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSS 156

Polynucleotides and protein expression methods

[00224] The invention also relates to nucleic acids (for example, DNA or RNA) encoding polypeptides of the present invention (for example, 5T4-binding polypeptides, 4-1BB-binding polypeptides and/or polyspecific binding proteins containing them) or one or more polypeptides polypeptide chains as described herein. The nucleic acids of the invention include nucleic acids having a region substantially identical to a polynucleotide listed in Tables 1-10 below. In certain embodiments, the nucleic acid of the present invention has at least 80%, typically at least about 90%, and more typically at least about 95%, or at least about 98% identity to a polynucleotide encoding a polypeptide listed in Tables 1 -10, where the nucleic acid encodes a polypeptide that is a polyspecific polypeptide in the format scFv-Fc-scFv, and where the encoded polypeptide contains one or more of (1) a Y to F substitution in LCDR1 at position 99 V _L to 5T4; (2) replacing F with S in FR3 at position 148 V _L to 5T4; (3) Y to H substitution in HCDR1 at position 150 V _H to 4-1BB; and (4) replacing R with H in FR3 at position 127 V _H to 4-1BB. Nucleic acids according to the invention also include complementary nucleic acids. In some cases, sequences will be completely complementary (no mismatches) when aligned. In other cases, the sequences may differ by up to approximately 20%. In some embodiments, the invention provides nucleic acids encoding both the first and second polypeptide chains of a bispecific protein of the invention. The nucleic acid sequences provided herein can be used using codon optimization, degenerate sequences, silent mutations and other DNA techniques to optimize expression in a particular host, and the present invention includes such sequence modifications.

[00225] The invention relates to an isolated nucleic acid molecule encoding polypeptides according to the present invention (for example, 5T4-binding polypeptides, 4-1BB-binding polypeptides and/or polyspecific binding proteins containing them), wherein said nucleic acid molecule contains a nucleotide sequence selected from SEQ ID NO: 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153 , 155, 171, 173 and 175.

[00226] Polynucleotide molecules containing the desired polynucleotide sequence are reproduced by placing the molecule in a vector. Viral and non-viral vectors, including plasmids, are used. The choice of plasmid will depend on the type of cell in which reproduction is desired and the purpose of reproduction. Some vectors are used to amplify and produce large quantities of the desired DNA sequence. Other vectors are suitable for expression in cells in culture. Other vectors are suitable for transfer and expression into cells in an animal or human in general. The selection of a suitable vector is within the skill of the art. Many such vectors are commercially available. Partial or full-length polynucleotide is usually inserted into a vector by attaching a DNA ligase to a cleavage restriction site in the vector. Alternatively, the desired nucleotide sequence can be inserted by in vivo homologous recombination. This is usually accomplished by joining regions of homology to the vector along the edges of the desired nucleotide sequence. Regions of homology are added by ligation of oligonucleotides or, for example, by polymerase chain reaction using primers containing both the region of homology and part of the desired nucleotide sequence.

[00227] An expression cassette or expression system may be used for expression. To express a nucleic acid encoding a polypeptide disclosed herein, a nucleic acid molecule encoding the polypeptide operably linked to regulatory sequences that control transcriptional expression in an expression vector is introduced into a host cell. In addition to transcriptional regulatory sequences such as promoters and enhancers, expression vectors may include translational regulatory sequences and a marker gene suitable for selecting cells harboring the expression vector. The gene product encoded by the polynucleotide of the invention is expressed in any suitable expression system, including, for example, a bacterial, yeast, insect, amphibian and mammalian system. In an expression vector, a polynucleotide encoding a polypeptide is linked to a regulatory sequence in an appropriate manner to obtain the desired expression properties. Such sequences may include promoters, enhancers, terminators, operators, repressors and inducers. Promoters can be regulated (eg, the promoter from the steroid-inducible vector pIND (Invitrogen)) or constitutive (eg, promoters from CMV, SV40, elongation factor, or LTR sequences). They are linked to the desired nucleotide sequence using the methods described above for linking to vectors. Any methods known in the art may be used. Accordingly, the expression vector will typically provide a transcriptional and translational initiation region, which may be inducible or constitutive, wherein the coding region is operably linked under transcriptional control to a transcriptional initiation region and a transcriptional and translational termination region.

[00228] The expression cassette ("expression unit") can be introduced into various vectors, for example, plasmid, BAC, YAC, bacteriophage such as lambda, P1, M13, etc., plant or animal virus vectors (e.g. , retrovirus-based vectors, adenoviral vectors) and the like, where the vectors are typically characterized by the ability to select cells containing the expression vectors. Vectors can provide maintenance in an extrachromosomal state, especially in the form of plasmids or viruses, or integration into the host chromosome. If extrachromosomal maintenance is required, an origin of replication sequence is provided for plasmid replication, which can be low or high copy number. There are a large number of different markers available to choose from, particularly those that provide protection against toxins, particularly antibiotics. The choice of a particular marker depends on the nature of the host, where complementation may be used in some cases with auxotrophic hosts. Any convenient method may be used to introduce the DNA construct, including, for example, conjugation, bacterial transformation, DNA calcium precipitation, electroporation, fusion, transfection, infection with viral vectors, biolistic transfection, and the like. The invention relates to an expression vector containing a nucleic acid segment, where the specified nucleic acid segment may contain a nucleotide sequence selected from SEQ ID NO: 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131 , 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 171, 173 and 175.

[00229] Accordingly, proteins for use in the present invention can be produced in genetically engineered host cells in accordance with conventional methods. Suitable host cells refer to cell types that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells and cultured cells of higher eukaryotes (including cultured cells of multicellular organisms), in particular cultured mammalian cells. Methods for manipulating cloned DNA molecules and introducing exogenous DNA into various host cells are described by Sambrook and Russell, Molecular Cloning: A Laboratory Manual (3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001), and Ausubel et al. , Short Protocols in Molecular Biology (4th ed., John Wiley & Sons, 1999).

[00230] For example, for the recombinant expression of a homodimeric binding protein containing two identical binding polypeptides, as described herein, the expression vector will typically include a nucleic acid segment encoding the binding polypeptide operably linked to a promoter. For recombinant expression of a heterodimeric binding protein containing different first and second polypeptide chains, the first and second polypeptide chains can be co-expressed from separate vectors in a host cell to express the entire heterodimeric protein. Alternatively, to express heterodimeric binding proteins, the first and second polypeptide chains are co-expressed from separate expression units in the same vector in a host cell to express the entire heterodimeric protein. The expression vector(s) are transferred into a host cell by conventional methods, and then the transfected cells are cultured by conventional methods to produce the encoded polypeptide(s) to produce the corresponding binding proteins (eg, 5T4-binding polypeptides, 4-1BB-binding polypeptides and/or containing their polyspecific binding proteins).

[00231] To direct the recombinant protein to the secretory pathway of the host cell, a secretory signal sequence (also known as a leader sequence) is provided in the expression vector. The secretory signal sequence may be that of the native form of the recombinant protein, or may be derived from another secreted protein, or synthesized de novo . Said secretory signal sequence is operably linked to a DNA sequence encoding the polypeptide, that is, the two sequences are joined in the correct reading frame and positioned to direct the newly synthesized polypeptide into the secretory pathway of the host cell. Secretory signal sequences are typically 5' terminal to the DNA sequence encoding the polypeptide of interest, although some signal sequences may be located elsewhere in the DNA sequence of interest (see, for example, US Pat. Nos. 5,037,743 and 5,143,830).

[00232] Cultured mammalian cells are suitable hosts for the production of recombinant polypeptides and proteins of the present invention (eg, 5T4-binding polypeptides, 4-1BB-binding polypeptides and/or polyspecific binding proteins containing them) for use in the present invention. Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection (Wigler et al. , Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981: Graham and Van der Eb, Virology 52: 456, 1973), electroporation (Neumann et al., EMBO J. 1:841-845, 1982), DEAE-dextran-mediated transfection (Ausubel et al., supra) and liposome-mediated transfection (Hawley-Nelson et al. , Focus 15:73, 1993; Ciccarone et al. , Focus 15:80, 1993). The production of recombinant polypeptides in cultured mammalian cells is disclosed, for example, in US patent Nos. 4,713,339; 4,784,950; 4,579,821 and 4,656,134. Examples of suitable mammalian host cells include African green monkey kidney cells (Vero; ATCC CRL 1587), human embryonic kidney cells (293-HEK; ATCC CRL 1573), neonatal hamster kidney cells (BHK-21, BHK-570; ATCC CRL 8544 , ATCC CRL 10314), canine kidney cells (MDCK; ATCC CCL 34), Chinese hamster ovary cells (CHO-K1; ATCC CCL61; CHO DG44; CHO DXB11 (Hyclone, Logan, UT); see also, e.g., Chasin et al., Som. Cell. Molec. Genet. 12:555, 1986)), rat pituitary cells (GH1; ATCC CCL82), HeLa S3 cells (ATCC CCL2.2), rat hepatoma cells (H-4-II-E ; ATCC CRL 1548), SV40-transformed monkey kidney cells (COS-1; ATCC CRL 1650) and mouse embryonic cells (NIH-3T3; ATCC CRL 1658). Other suitable cell lines are known in the art and are available from public repositories such as the American Type Culture Collection, Manassas, Virginia. Strong transcription promoters, such as those from SV-40 or cytomegalovirus, can be used. See, for example, US Patent No. 4,956,288. Other suitable promoters include those from the metallothionein genes (US Patent Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.

[00233] Drug sensitivity selection is commonly used to select cultured mammalian cells into which foreign DNA has been inserted. Such cells are usually called "transfectants". Cells cultured in the presence of a selective agent and capable of passing on the gene of interest to their progeny are called “stable transfectants.” Exemplary selectable markers include a gene encoding resistance to the antibiotic neomycin, which allows selection in the presence of a neomycin-type drug such as G-418 or the like; xanthine-guanine-phosphoribosyltransferase gene gpt, which promotes growth of host cells in the presence of mycophenolic acid/xanthine; and markers conferring resistance to zeocin, bleomycin, blasticidin and hygromycin (see, for example, Gatignol et al., Mol. Gen. Genet. 207:342, 1987; Drocourt et al., Nucl. Acids Res . 18:4009, 1990). Selection systems can also be used to increase the expression level of a gene of interest, a process called "amplification". Amplification is accomplished by culturing the transfectants in the presence of a low level of the selection agent and then increasing the amount of the selection agent to select for cells producing high levels of the introduced gene products. An exemplary amplifiable selectable marker is a dihydrofolate reductase conferring resistance to methotrexate. Other genes that confer drug resistance (eg, hygromycin resistance, multidrug resistance, puromycin acetyltransferase) may also be used.

[00234] Other higher eukaryotic cells can also be used as hosts, including insect cells, plant cells and avian cells. The use of Agrobacterium rhizogenes as a vector for gene expression in plant cells was reviewed by Sinkar et al. , J. Biosci. (Bangalore) 11:47-58, 1987. Transformation of insect cells and production of foreign polypeptides therein are disclosed in US Patent No. 5,162,222 and PCT Publication No. WO 94/06463.

[00235] Insect cells can be infected with a recombinant baculovirus, typically derived from Autographa californica nuclear polyhedrosis virus (AcNPV). See King and Possee, The Baculovirus Expression System: A Laboratory Guide (Chapman & Hall, London); O'Reilly et al. , Baculovirus Expression Vectors: A Laboratory Manual (Oxford University Press., New York 1994); and Baculovirus Expression Protocols. Methods in Molecular Biology (Richardson ed., Humana Press, Totowa, NJ, 1995). Recombinant baculovirus can also be produced by using the transposon-based system described by Luckow et al. ( J. Virol. 67:4566-4579, 1993). This system, which uses transfer vectors, is commercially available in kit form (BAC-TO-BAC kit; Life Technologies, Gaithersburg, MD). The transfer vector (eg, PFASTBAC1; Life Technologies) contains the Tn7 transposon to move the DNA encoding the protein of interest into the baculovirus genome, maintained in E. coli as a large plasmid called a “bacmid.” see Hill-Perkins and Possee, J. Gen. Virol. 71:971-976, 1990; Bonning et al. , J.Gen. Virol. 75:1551-1556, 1994; and Chazenbalk and Rapoport, J. Biol. Chem. 270:1543-1549, 1995. In addition, transfer vectors can include an in-frame fusion to DNA encoding a polypeptide extension or affinity tag, as described above. Using methods known in the art, a transfer vector containing a DNA sequence encoding a protein is transformed into E. coli host cells, and the cells are screened for the presence of bacmids containing an interrupted lacZ gene, indicative of a recombinant baculovirus. Bacmid DNA containing the recombinant baculovirus genome is isolated using conventional methods and used to transfect Spodoptera frugiperda cells, such as Sf9 cells. A recombinant virus expressing the protein of interest is then produced. The original recombinant viruses are prepared by methods commonly used in the art.

[00236] To produce the protein, the recombinant virus is used to infect host cells, typically a cell line derived from the armyworm, Spodoptera frugiperda (eg, Sf9 or Sf21 cells) or Trichoplusia ni (eg, HIGH FIVE™ cells; Invitrogen, Carlsbad , California). For an overview, see Glick and Pasternak, Molecular Biotechnology, Principles & Applications of Recombinant DNA (ASM Press, Washington, DC, 1994) . See also US Patent No. 5,300,435. Serum-free media is used to grow and maintain cells. Suitable media compositions are known in the art and can be obtained from commercial suppliers. Cells are grown from an inoculation density of approximately 2-5 x 10 ⁵ cells to a density of 1-2 x 10 ⁶ cells, at which time the parent recombinant virus is added at a multiplicity of infection (MOI) of 0.1 to 10, typically approximately 3. The procedures used are usually described in available laboratory manuals (see, for example, King and Possee, cited above; O'Reilly et al. , cited above; Richardson, cited above).

[00237] Fungal cells, including yeast cells, can also be used in the present invention to produce the polypeptides of the present invention (eg, 5T4-binding polypeptides, 4-1BB-binding polypeptides and/or polyspecific binding proteins containing them). Suitable yeast species include, for example, Saccharomyces cerevisiae , Pichia pastoris and Pichia methanolica . Methods for transforming S. cerevisiae cells with exogenous DNA and obtaining recombinant polypeptides from them are described, for example, in US patents No. 4,599,311; 4,931,373; 4,870,008; 5,037,743; and 4,845,075. Transformed cells are selected for a phenotype defined by a selectable marker, typically drug resistance or the ability to grow in the absence of a particular nutrient (eg, leucine). An exemplary vector system for use in Saccharomyces cerevisiae is the POT1 vector system disclosed by Kawasaki et al. (US Patent No. 4,931,373), which allows the selection of transformed cells by growing in media containing glucose. Suitable promoters and terminators for use in yeast include those from the glycolytic enzyme genes (see, for example, US Pat. Nos. 4,599,311; 4,615,974 and 4,977,092) and alcohol dehydrogenase genes. See also US Patent Nos. 4,990,446; 5,063,154; 5,139,936 and 4,661,454. Transformation systems for other yeasts are known in the art, including Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichia guillermondii and Candida maltosa . See, for example, Gleeson et al. , J.Gen. Microbiol. 132:3459-3465, 1986; US Patent No. 4,882,279; and Raymond et al. , Yeast 14:11-23, 1998. Aspergillus cells can be used according to methods according to McKnight et al. , US Patent No. 4,935,349. Methods for transforming Acremonium chrysogenum are disclosed by Sumino et al. , US Patent No. 5,162,228. Methods for transforming Neurospora are disclosed by Lambowitz, US Patent No. 4,486,533. The production of recombinant proteins in Pichia methanolica is described in US Patent Nos. 5,716,808; 5,736,383; 5,854,039 and 5,888,768.

[00238] Prokaryotic host cells, including strains of bacteria Escherichia coli , Bacillus and other genera, are also suitable for use as host cells within the scope of the present invention for the production of, for example, 5T4-binding polypeptides, 4-1BB-binding polypeptides and/ or polyspecific binding proteins containing them, including molecules to 5T4 x 4-1BB. Methods for transforming these hosts and expressing foreign DNA sequences cloned into them are well known in the art (see, for example, Sambrook and Russell, cited above). When a recombinant protein is expressed in bacteria such as E. coli , the protein may be retained in the cytoplasm, usually as insoluble granules, or may be directed into the periplasmic space through a bacterial secretion sequence. In the first case, the cells are lysed and the granules are isolated and denatured using, for example, guanidine isothiocyanate or urea. The denatured protein can then be refolded and dimerized by diluting the denaturing agent, for example by dialysis against a solution of urea and a combination of reduced and oxidized glutathione, followed by dialysis against buffered saline. Alternatively, the protein can be recovered from the cytoplasm in soluble form and isolated without the use of denaturing agents. The protein is extracted from the cell in the form of an aqueous extract, for example, in sodium phosphate buffer. To capture the protein of interest, the extract is applied directly to a chromatographic medium, such as an immobilized antibody or a heparin-Sepharose column. Secreted proteins can be recovered from the periplasmic space in a soluble and functional form by disrupting cells (e.g., ultrasound or osmotic shock) to release the contents of the periplasmic space and release the protein, thereby eliminating the need for denaturation and refolding. Antibodies, including single chain antibodies, can be produced in bacterial host cells according to known methods. See, for example, Bird et al. , Science 242:423-426, 1988; Huston et al. , Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988; and Pantoliano et al. , Biochem . 30:10117-10125, 1991.

[00239] Transformed or transfected host cells to produce the polypeptides and proteins of the present invention (e.g., 5T4-binding polypeptides, 4-1BB-binding polypeptides, and/or polyspecific binding proteins containing them) are cultured according to conventional techniques in a culture medium, containing nutrients and other components necessary for the growth of selected host cells. Various suitable media are known in the art, including formulated media and complex media, and typically include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. The media may also optionally contain components such as growth factors or serum. The culture medium typically allows for the selection of cells containing exogenously added DNA, for example, by selection for drug sensitivity or deficiency of an essential nutrient, which is supplemented by a selectable marker carried on an expression vector or co-transfected into the host cell.

[00240] The proteins and polypeptides of the present invention (eg, 5T4-binding polypeptides, 4-1BB-binding polypeptides and/or polyspecific binding proteins containing them) can be purified by conventional protein purification methods, typically using a combination of chromatographic techniques. For a general overview, see Affinity Chromatography: Principles & Methods (Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988); Scopes, Protein Purification: Principles and Practice (Springer-Verlag, New York 1994). Proteins containing the immunoglobulin Fc region can be purified by affinity chromatography on immobilized protein A or protein G. Additional purification steps, such as gel filtration, can be used to obtain the desired level of purity or for desalting, buffer exchange, and the like.

Compositions and methods of use

[00241] The present invention relates to methods of treating a subject suffering from a disorder characterized by 5T4 expression. Typically, such methods involve administering to a subject in need of such treatment a protein of the present invention (eg, 5T4 binding polypeptides, 4-1BB binding polypeptides, and/or polyspecific binding proteins containing them). In some embodiments, the 5T4-binding polypeptide, 4-1BB-binding polypeptide, and/or polyspecific binding proteins containing them (e.g., a 5T4 x 4-1BB molecule) induce no or minimal antibody-dependent cell-mediated cytotoxic activity (ADCC) and/or or complement dependent cytotoxic activity (CDC).

[00242] In other embodiments, where the binding proteins (e.g., polyspecific binding proteins) contain a second binding domain that specifically binds an effector cell (e.g., 4-1BB), the binding proteins and/or polyspecific binding proteins result in enhanced activation of effector cells against 5T4-expressing cells in the subject. For example, in some embodiments, binding proteins and/or multispecific binding proteins lead to increased proliferation of effector cells, increased production of one or more cytokines by effector cells (e.g., IFNγ, TNFα, IL-6, IL-8, IL-12, IL- 1α, etc.) or increased expression of one or more cell surface activation markers (eg CD137, MHC-II or CD69). Effector cell activation can be measured by a variety of methods known in the art, including flow cytometry, immunofluorescence and immunohistochemistry to assess changes in the expression of cell surface markers, ELISA to assess the production of cytokines and other factors, cell counting methods to assess proliferation, qPCR to assessing changes in gene expression and the like.

[00243] In some embodiments, the polyspecific binding proteins described herein exhibit increased activation of effector cells compared to a second polyspecific polypeptide of a different structure. For example, in some embodiments, the first polyspecific polypeptide comprises a first scFv domain that specifically binds to 5T4 and a second scFv that specifically binds to 4-1BB, linked together through a binding domain linker, and an immunoglobulin Fc domain in the following configuration, in a direction away from amino terminus to carboxy terminus: (i) first scFv domain, (ii) hinge region, (iii) immunoglobulin constant region, (iv) linker binding domains and (v) second scFv domain. In this example, the second multispecific binding protein contains an IgG-scFv structure containing an anti-4-1BB antibody and an anti-5T4 scFv. In some embodiments, the first polyspecific binding protein exhibits a statistically significant increase in effector cell activation compared to the second polyspecific polypeptide. For example, in some embodiments, the first polyspecific binding protein exhibits a lower EC ₅₀ value in a Jurkat/NF-κB reporter cell assay than is observed for the second polyspecific binding protein (see, for example, Example 10). In some embodiments, the EC ₅₀ of the first polyspecific binding protein is reduced by about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold , approximately 10 times or more compared to the EC ₅₀ of the second polyspecific binding protein. EC ₅₀ values can be determined using nonlinear regression analysis and other statistical methods known in the art.

[00244] In some embodiments, the first polyspecific binding protein induces a statistically significant increase in effector cell proliferation compared to the second polyspecific polypeptide. For example, in some embodiments, the first polyspecific binding protein induces a statistically significant increase in the proliferation of primed human CD8+ T cells compared to the proliferation induced by the second polyspecific binding protein (see, for example, Example 22). In some embodiments, the first multispecific binding protein induces an increase in effector cell proliferation of about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, approximately 10-fold or more, the effector cell proliferation induced by the second polyspecific binding protein.

[00245] In certain variations of a method of treating a subject with a protein of the present invention (eg, 5T4 binding polypeptides, 4-1BB binding polypeptides, and/or polyspecific binding proteins containing them), the disorder is cancer. Exemplary cancers amenable to treatment with the protein of the present invention include, for example, breast cancer (eg, triple negative breast cancer (TNBC)), pancreatic cancer, ovarian cancer, lung cancer (eg, non-small cell lung cancer), hematologic malignancies ( eg, chronic lymphocytic leukemia (CLL), mantle cell leukemia (MCL) or acute lymphoblastic leukemia (ALL)), skin cancer (eg, squamous cell carcinoma or melanoma), adrenal cancer, bladder cancer, cervical cancer, kidney cancer, stomach cancer , prostate cancer, thyroid cancer, liver cancer, uterine cancer, a tumor formed on neurons or the sheath of nerve fibers (for example, neurofibroma), sarcoma or cancer of the head and neck. TNBC is defined as breast cancer lacking staining for estrogen receptor, progesterone receptor, and HER2/neu . In some embodiments, a protein of the present invention may be administered to a subject to treat said subject's mesothelioma. In one embodiment, a protein of the present invention may be administered to a subject to treat said subject's clear cell carcinoma. In one embodiment, a protein of the present invention may be administered to a subject to treat said subject's striated muscle tumor.

[00246] In a further embodiment, the invention includes a method of enhancing effector cell activation against a 5T4-expressing cell, comprising contacting said 5T4-expressing cell with a protein of the present invention (e.g., 5T4-binding polypeptides, 4-1BB-binding polypeptides and/or polyspecific binding proteins containing them), wherein said contacting is carried out under conditions under which enhanced activation of effector cells directed against said 5T4-expressing cell is induced. In some embodiments, the invention provides a method of enhancing effector cell activation directed against a 5T4-expressing cell, comprising contacting said 5T4-expressing cell with a protein of the present invention (e.g., 5T4-binding polypeptides, 4-1BB-binding polypeptides, and/or or polyspecific binding proteins containing them) comprising a first binding domain that specifically binds a human 5T4 epitope and a second binding domain that specifically binds 4-1BB (eg, a 5T4 x 4-1BB molecule); wherein said contacting is carried out under conditions that induce enhanced activation of effector cells directed against said 5T4-expressing cell. The invention includes a method of inducing effector cell-dependent lysis of a 5T4-expressing cell, comprising contacting said 5T4-expressing cell with a protein of the present invention (e.g., 5T4-binding polypeptides, 4-1BB-binding polypeptides, and/or polyspecific binding proteins containing them ), where the second binding domain specifically binds 4-1BB (eg, a molecule to 5T4 x 4-1BB); and wherein said contacting is carried out under conditions under which enhanced activation of effector cells is induced against said 5T4-expressing cell, resulting in lysis of said 5T4-expressing cell. In some embodiments, the invention provides a method for inducing effector cell-dependent lysis of a 5T4-expressing cell, comprising contacting said 5T4-expressing cell with a protein of the present invention comprising a first binding domain that specifically binds an epitope of human 5T4, and a second binding domain, specifically binding 4-1BB (for example, a molecule to 5T4 x 4-1BB); wherein said contacting is carried out under conditions under which enhanced activation of effector cells is induced against said 5T4-expressing cell, resulting in lysis of said 5T4-expressing cell.

[00247] The invention also includes proteins and polypeptides (eg, 5T4-binding polypeptides, 4-1BB-binding polypeptides and/or polyspecific binding proteins containing them) for the production of a medicament for the treatment of a disorder (eg, cancer) characterized by 5T4 expression. In one embodiment, the protein or polypeptide comprises a 5T4 and 4-1BB binding domain (eg, a 5T4 x 4-1BB molecule) and has enhanced effector cell activation activity. In one embodiment, the invention provides proteins and polypeptides (eg, a 5T4 x 4-1BB molecule) for use in treating a disorder (eg, cancer) characterized by 5T4 expression. In certain embodiments, the invention provides a method of treating a disorder in a subject, wherein said disorder is characterized by expression of 5T4, comprising administering to said subject a therapeutically effective amount of a protein or polypeptide of the present invention comprising a 5T4-binding domain that specifically binds an epitope of human 5T4 (e.g., 5T4- binding polypeptides, 4-1BB-binding polypeptides and/or polyspecific binding proteins containing them).

[00248] In some embodiments, the invention provides a method of treating a patient suffering from cancer, comprising administering to said patient a polypeptide comprising an amino acid sequence provided herein (e.g., an amino acid sequence selected from the group consisting of SEQ ID NO: 136, 138 , 140, 142, 144, 146, 148, 150, 152, 154, 156, 172, 174 and 176). In additional embodiments, the polypeptide comprises an Fc. For example, in some embodiments, the present invention provides a method of treating a patient suffering from cancer, comprising administering to said patient a polypeptide comprising a 5T4 binding domain and an Fc, wherein said polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 172, 174, and 176. In further embodiments, the present invention provides a method of treating a patient suffering from cancer, comprising administering to said patient a polypeptide containing 5T4- binding domain and a 4-1BB binding domain, wherein said polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 172 , 174 and 176. In one embodiment, the present invention provides a method of treating a patient suffering from cancer, comprising administering to said patient a polypeptide comprising a 5T4 binding domain and a 4-1BB binding domain, wherein said polypeptide comprises SEQ ID NO: 172. In one embodiment, the present invention provides a method of treating a patient suffering from cancer, comprising administering to said patient a polypeptide comprising a 5T4 binding domain and a 4-1BB binding domain, wherein said polypeptide comprises SEQ ID NO: 174.

[00249] In some embodiments, for the treatments and uses described herein, a protein or polypeptide described herein (e.g., 5T4 binding polypeptides, 4-1BB binding polypeptides, and/or polyspecific binding proteins containing them) delivered in a manner consistent with generally accepted principles associated with the control of the disease or disorder being treated. As described herein, a therapeutically effective amount of a polypeptide protein is administered to a subject in need of such treatment for a time and under conditions sufficient to prevent or treat the disease or disorder.

[00250] Subjects to be administered with the protein of the present invention (e.g., 5T4 binding polypeptides, 4-1BB binding polypeptides, and/or polyspecific binding proteins containing them) include patients at high risk for developing a particular disorder characterized by 5T4 expression, as well as patients already suffering from such a disorder. Typically, the subject has been diagnosed with a disorder to be treated. In addition, subjects may be monitored during treatment for any change in the disorder (eg, increase or decrease in clinical symptoms of the disorder). Additionally, in some variations, the subject does not suffer from another disorder requiring treatment associated with targeting the 5T4-expressing cells.

[00251] For prophylactic purposes, pharmaceutical compositions or drugs containing a protein according to the present invention (for example, 5T4-binding polypeptides, 4-1BB-binding polypeptides and/or polyspecific binding proteins containing them) are administered to a patient who is vulnerable or otherwise at risk development of a specified disorder, in an amount sufficient to eliminate or reduce the risk of or delay the occurrence of the specified disorder. For therapeutic purposes, compositions or drugs containing the protein of the present invention are administered to a patient suspected of suffering or already suffering from such a disorder in an amount sufficient to cure or at least partially resolve the symptoms of the disorder and its complications. An amount sufficient to achieve this purpose is called a therapeutically effective dose or amount. In both prophylactic and treatment regimens, agents are typically administered in multiple doses until a sufficient response is achieved (eg, inhibition of abnormal angiogenic activity). Typically, the response is monitored and repeat doses are given if the desired response begins to wane.

[00252] To identify patients to be treated with the methods of the invention (e.g., 5T4-binding polypeptides, 4-1BB-binding polypeptides and/or polyspecific binding proteins containing them), established screening methods can be used to determine risk factors associated with specific disorders, or to determine the status of an existing disorder identified in a subject. Such methods may include, for example, determining whether an individual has relatives who have been diagnosed with a particular disease. Screening methods may also include, for example, routine examinations to determine the familial status of a particular disease known to have a hereditary component. For example, different types of cancer are known to have certain hereditary components. Hereditary components of cancers include, for example, transforming mutations in several genes (eg, Ras, Raf, EGFR, cMet, etc.), the presence or absence of individual human leukocyte antigen (HLA) and cytotoxicity inhibitory receptor (KIR) molecules, or mechanisms by which cancer cells are able to modulate the suppression of the immune response of cells such as NK cells and T cells, directly or indirectly (see, for example, Ljunggren and Malmberg, Nature Rev. Immunol. 7:329-339, 2007; Boyton and Altmann , Clin. Exp. Immunol. 149:1-8, 2007). To this end, in accordance with standard practice, nucleotide probes can be used to identify individuals carrying genetic markers associated with a particular disorder of interest. In addition, a variety of immunological methods are known in the art that are suitable for identifying markers of a particular disorder. For example, various ELISA methods that use monoclonal antibody probes to detect antigens associated with specific tumors are available and well known in the art. Screening may be performed according to the patient's known symptoms, age factors, associated risk factors, and the like. These methods allow the attending physician to select patients in need of the treatments described herein in a standardized manner. In accordance with these methods, targeting pathological cells expressing 5T4 can be implemented as an independent treatment program or as a subsequent, supportive or coordinating treatment regimen to other treatments.

[00253] For administration, the protein of the present invention (eg, 5T4-binding polypeptides, 4-1BB-binding polypeptides and/or polyspecific binding proteins containing them) can be formulated into a pharmaceutical composition. The pharmaceutical composition may contain: (i) a 5T4 binding polypeptide, a 4-1BB binding polypeptide and/or a polyspecific binding protein containing them (eg, a 5T4 x 4-1BB molecule); and (ii) a pharmaceutically acceptable carrier, diluent or excipient. A pharmaceutical composition comprising a 5T4 binding polypeptide, a 4-1BB binding polypeptide and/or a polyspecific binding protein containing them (for example, a 5T4 x 4-1BB molecule) can be prepared in accordance with known methods for the preparation of pharmaceutically acceptable compositions, in which the therapeutic molecule is combined in admixture with a pharmaceutically acceptable carrier, diluent or excipient. A carrier is considered to be a “pharmaceutically acceptable carrier” if its administration is tolerated by the recipient patient. Sterile sodium phosphate buffer is one example of a pharmaceutically acceptable carrier. Other suitable carriers, diluents or auxiliaries are well known to those skilled in the art. (See, e.g., Gennaro (ed.), Remington's Pharmaceutical Sciences (Mack Publishing Company, 19th ed. 1995).) The compositions may further include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent loss of protein by surfaces of ampoules, etc. In certain embodiments, the pharmaceutical composition contains a 5T4-binding polypeptide, a 4-1BB-binding polypeptide and/or a polyspecific binding protein containing them (for example, a molecule to 5T4 x 4-1BB), which is a homodimer or heterodimer . A "homodimer" may be a dimer formed from two identical polypeptides (eg, a 5T4 x 4-1BB molecule as described herein).

[00254] A pharmaceutical composition containing a polypeptide or protein described herein may be provided in a dosage form selected from the group consisting of: single dose oral form, single dose intravenous form, single dose intranasal form, single dose suppository form, single dose intradermal form, single dose intramuscular form, single dose intraperitoneal form, single dose subcutaneous form, single dose epidural form, single dose sublingual form and single dose intracerebral form. The single dose oral form may be selected from the group consisting of tablets, pills, pellets, capsules, powders, lozenges, granules, solutions, suspensions, emulsions, syrups, elixirs, sustained release formulations, aerosols and sprays.

[00255] A pharmaceutical composition containing a polypeptide or protein described herein (eg, 5T4 binding polypeptides, 4-1BB binding polypeptides, and/or polyspecific binding proteins containing them) can be administered to a subject in a therapeutically effective amount. In accordance with the methods of the present invention, the polypeptide or protein described herein (e.g., 5T4-binding polypeptides, 4-1BB-binding polypeptides, and/or polyspecific binding proteins containing them) can be administered to subjects by various routes of administration, including, for example , intramuscular, subcutaneous, intravenous, intraatrial, intraarticular, parenteral, intranasal, intrapulmonary, percutaneous, intrapleural, intrathecal and oral routes of administration. For purposes of prevention and treatment, an agonist (eg, 5T4 binding polypeptides, 4-1BB binding polypeptides, and/or polyspecific binding proteins containing them) may be administered to a subject via a single bolus delivery, via continuous delivery (eg, continuous transdermal delivery) over over a long period of time, or on a repeated dosing schedule (eg, hourly, daily, weekly, or monthly).

[00256] Determination of effective dosages in this context is typically based on animal model studies followed by human clinical studies, and is determined according to effective dosages and administration schedules that significantly reduce the incidence or severity of the disorder being treated in the model subjects. Effective dosages of the compositions of the present invention vary depending on many different factors, including the route of administration, the target site, the physiological state of the patient, whether the patient is human or animal, other drugs administered, whether the treatment is prophylactic or therapeutic, and the specific activity of the composition itself. and its ability to elicit a desired response in an individual. Typically the patient is human, but in some diseases the patient may be a non-human mammal. Typically, dosing regimens are adjusted to ensure optimal therapeutic response, that is, to optimize safety and efficacy. Accordingly, a therapeutically effective amount is also one such that any undesirable side effects are outweighed by the beneficial effects of administration of the 5T4 binding polypeptide, 4-1BB binding polypeptide, and/or a polyspecific binding protein containing them (e.g., 5T4 x 4-1BB molecule ) as described herein. For administration of a protein or polypeptide described herein, the dosage may be from about 0.1 μg to 100 mg/kg, or from 1 μg/kg to about 50 mg/kg, and more commonly from 10 μg to 5 mg/kg of the subject's body weight . In particular embodiments, the effective amount of the agent is from about 1 μg/kg to about 20 mg/kg, from about 10 μg/kg to about 10 mg/kg, or from about 0.1 mg/kg to about 5 mg/kg. Dosages in this range can be achieved by single or multiple administrations, including, for example, multiple administrations several times a day or daily, weekly, biweekly or monthly. For example, in some variations, the regimen consists of an initial administration followed by multiple sequential administrations at weekly or biweekly intervals. Another regimen consists of an initial administration followed by multiple sequential administrations at monthly or bimonthly intervals. Alternatively, administration may be administered at irregular intervals, depending on the observed clinical symptoms of the disorder.

[00257] The dosage of a pharmaceutical composition containing a polypeptide or protein described herein (e.g., 5T4-binding polypeptides, 4-1BB-binding polypeptides, and/or polyspecific binding proteins containing them) may be varied by the attending physician to maintain the desired concentration at the target website. For example, if an intravenous delivery route is selected, the local concentration of the agent in the bloodstream in the target tissue may be from about 0.01 to 50 nanomoles of the composition per liter, sometimes from about 1.0 nanomoles per liter to 10, 15, or 25 nanomoles per liter, depending on the subject's condition and the predicted measured response. Higher or lower concentrations may be selected depending on the mode of delivery, eg, transepidermal versus mucosal delivery. Dosage should also be adjusted based on the rate of release of the drug administered, e.g., nasal spray versus powder, oral versus injectable sustained release particles, transdermal drugs, etc. For example, twice the serum concentration is required to achieve the same level of serum concentration. higher dosing of slow release particles with a release rate of 5 nanomolar units (under standard conditions) compared to dosing of particles with a release rate of 10 nanomolar units.

[00258] Proteins and polypeptides described herein (e.g., 5T4 binding polypeptides, 4-1BB binding polypeptides, and/or polyspecific binding proteins containing them) (e.g., 5T4 x 4-1BB molecule) may also be administered at a daily dose of about 0.001 to about 10 milligrams (mg) per kilogram (mg per kg) of body weight, preferably as a single daily dose or in divided doses from about two to six times daily. For administration to an adult human patient, a therapeutically effective amount may be administered in doses ranging from 0.2 to 800 mg per dose, including, but not limited to, 0.2 mg per dose, 0.5 mg per dose, 1 mg per dose , 5 mg per dose, 10 mg per dose, 25 mg per dose, 100 mg per dose, 200 mg per dose, and 400 mg per dose, and multiple, usually sequential daily doses may be administered over the course of treatment. The proteins and polypeptides described herein (eg, 5T4 binding polypeptides, 4-1BB binding polypeptides and/or polyspecific binding proteins containing them, such as the 5T4 x 4-1BB molecule) can be administered at different times of the day. In one embodiment, the optimal therapeutic dose may be administered in the evening. In yet another embodiment, the optimal therapeutic dose may be administered in the morning. Thus, the total daily dose of the proteins and polypeptides described herein, in one embodiment, can be from about 1 mg to about 2 g, and often is from about 100 mg to about 1.5 g, and most often is from approximately 200 mg to approximately 1200 mg. For a typical 70 kg adult, the total daily dose of 5T4 therapeutic agent may range from about 2 mg to about 1200 mg and will often, as noted above, range from about 0.2 mg to about 800 mg.

[00259] For the treatment of solid tumors, protocols for assessing endpoints and antitumor activity are well known in the art. Although each protocol may define tumor response scores differently, the RECIST (Response Evaluation Criteria in Solid Tumors) criteria are currently considered the recommended guidelines for tumor response assessment by the National Cancer Institute (see Therasse et al. , J. Natl. Cancer Inst. 92:205-216, 2000). According to RECIST criteria, tumor response means reduction or elimination of all measurable lesions or metastases. Disease is generally considered measurable if it includes lesions that can be accurately measured in at least one direction as equal to or greater than 20 mm using conventional methods, or equal to or greater than 10 mm using helical computed tomography with well-defined boundaries, using medical photography or radiography, computed axial tomography (CT), magnetic resonance imaging (MRI), or clinical examination (if lesions are superficial). Immeasurable disease means that the disease includes lesions that are less than 20 mm in size as measured by conventional methods or less than 10 mm as measured by helical computed tomography, and fundamentally immeasurable lesions (too small to be measured accurately). Unmeasurable disease includes pleural effusions, ascites, and circumstantial evidence-based disease.

[00260] Objective status criteria are required for protocols for assessing solid tumor response. Illustrative criteria include the following: (1) complete response (CR), defined as complete resolution of all measurable disease; absence of new lesions; absence of disease-related symptoms; lack of evidence of unmeasured disease; (2) partial response (PR), defined as a 30% reduction in the sum of the longest diameter of the target lesions; (3) progressive disease (PD), defined as a 20% increase in the sum of the longest diameter of the target lesions or the appearance of any new lesion; (4) stable or no response, defined as not meeting criteria for CR, PR, or progressive disease (see Therasse et al., cited above).

[00261] Additional endpoints that are accepted in the oncology field include overall survival (OS), disease-free survival (DFS), objective response rate (ORR), time to progression (TTP) and progression-free survival (PFS) (see Guidance for Industry: Clinical Trial Endpoints for the Approval of Cancer Drugs and Biologics , April 2005, Center for Drug Evaluation and Research, FDA, Rockville, MD.)

[00262] Pharmaceutical compositions containing the proteins and polypeptides described herein (e.g., 5T4 binding polypeptides, 4-1BB binding polypeptides, and/or polyspecific binding proteins containing them, such as the 5T4 x 4-1BB molecule) may supplied as a kit including a container containing a pharmaceutical composition as described herein. The pharmaceutical composition may be provided, for example, in the form of an injection solution for single or multiple doses or in the form of a sterile powder that must be reconstituted before injection. Alternatively, such a kit may include a dry powder nebulizer, a liquid aerosol generator, or a nebulizer for administering the pharmaceutical composition. Such a kit may further include written information regarding the indications and use of the pharmaceutical composition.

[00263] The invention is further illustrated by the following examples, which are intended solely to illustrate the invention and are not intended to be limiting in any way.

Examples

Example 1. Conversion of monoclonal antibody to CD137 “1618” into scFv format and production of bispecific molecules to CD137 x 5T4 in ADAPTIR™ format (scFv-Fc-scFv)

[00264] To generate the nucleotide sequence encoding scFv 1618, nucleotide sequences encoding the heavy chain ( _VH ) and light chain ( _VL ) variable domains (SEQ ID NO: 28 and SEQ ID NO: 16, respectively) from the monoclonal antibody to 4-1BB 1618 was synthesized and/or amplified from existing plasmid DNA and linked together using a (Gly) ₄ Ser based linker using standard molecular biology techniques described in, but not limited to, PCT Application Publications Nos. WO 2007/ 146968, WO 2010/040105 and WO 2010/003108; US Patent Application Publication No. 2006/0051844; and US Patent No. 7,166,707. The scFv 1618 nucleotide sequence was then fused to a modified human IgG1 Fc region sequence containing point mutations to eliminate the effector functional activity of the Fc region. Similarly, the nucleotide sequences encoding the V _H and V _L domains of the anti-5T4 monoclonal antibody 1210 (SEQ ID NOs: 38 and 68, respectively) were linked to each other by an additional (Gly) ₄ Ser-based linker. The resulting bispecific molecules were expressed by transiently transfecting HEK-293 cells or Chinese hamster ovary (CHO) cells and purified from conditioned media using protein A (ProA) affinity purification and size exclusion chromatography (SEC). Protein purity was determined by analytical SEC after each of the protein A and SEC purification steps. Endotoxin levels were determined using the Endosafe PTS instrument according to the manufacturer's instructions to ensure that the results of the in vitro potency assay were not confounded by the presence of endotoxin. Each protein batch was buffer-exchanged to PBS as part of the SEC purification process, concentrated to 1 mg/mL, subjected to sterilization filtration, and stored at 4°C until next use. Protein concentration was determined by absorbance at 280 nm using the theoretical extinction coefficient.

[00265] In some cases, bispecific molecules consisted of 2 scFv and 1 Fc domain (scFv-Fc-scFv) with the following structure, from N-terminus to C-terminus: scFv 1618 - Fc - scFv 1210. Additional bispecific molecules in which scFv 1618 was placed at the C-terminus of the construct and scFv 1210 at the N-terminus (i.e., scFv 1210 - Fc - scFv 1618). However, molecules with this alternative orientation had less desirable properties compared to scFv 1618 - Fc - scFv 1210-Fc-scFv molecules.

[00266] Most, but not all, of the 1618-Fc-scFv 1210-Fc-scFv ADAPTIR ^TM molecules (for example, ALG.APV-006, ALG.APV-010, ALG.APV-014 and ALG.APV-018) had significantly improved expression levels when transiently transfected into HEK-293 cells compared to the Morrison format (i.e., ALG.APV-004). Table 12 provides illustrative data demonstrating improved expression levels of ADAPTIR™ bispecific molecules (e.g., ALG.APV-006, ALG.APV-010, ALG.APV-014, and ALG.APV-018) compared to Morrison bispecific constructs (ALG .APV-004). This result was unexpected because the amino acid sequence of variable domains 1618 and 1210 was not modified to achieve this improvement. These results indicate that ADAPTIR ^™ bispecific molecules may be advantageous for the production of therapeutic protein drugs; Higher expression levels are generally considered an advantage in the production of therapeutic protein drugs, as it can provide lower cost of goods and other manufacturing process efficiencies, such as fewer production runs required to meet market demand.

[00267] In addition to the apparent improvement in expression in HEK-293, aggregate levels quantified after protein A purification of the bispecific molecule from conditioned media were significantly improved in the ADAPTIR ^™ bispecific constructs (ALG.APV-006, ALG.APV-010, ALG. APV-014 and ALG.APV-018) compared to the Morrison bispecific format (ALG.APV-004), as shown in Table 12.

Table 12. Transient Expression Levels in HEK-293 and Purification After Protein A Purification of ADAPTIR ^™ Bispecific Constructs Compared to Morrison Bispecific Constructs

Design Design Features Bispecific format Expression level, µg/ml % purity by analytical SEC method ALG.APV-004 mAb 1618-V _H V _L 1210 with a stabilizing disulfide bond Morrison 13.0 81.7 ALG.APV-006 V _H V _L 1618-Fc-V _H V _L 1210 ^ADAPTIRTM 25.5 94.4 ALG.APV-010 V _H V _L 1618-Fc-V _L V _H 1210 ^ADAPTIRTM 55.9 94.3 ALG.APV-014 V _H V _L 1618-Fc V _H V _L 1210 with stabilizing disulfide bond ^ADAPTIRTM 18.9 89.2 ALG.APV-018 V _H V _L 1618-Fc V _L V _H 1210 with stabilizing disulfide bond ^ADAPTIRTM 25.8 89.1

[00268] The order of the V _H and V _L domains was also assessed with both scFv 1210 and 1618 (V _H -V _L or V _L -V _H ), and the preferred orientation of scFv 1618 was determined to be V _H -V _L. since expression levels of the scFv-Fc-scFv molecule were significantly reduced when using the V _L -V _H orientation. The preferred orientation of scFv 1210 was V _H -V _L , which provided improved binding to 5T4-expressing cells and in vitro activity in the CD137 reporter assay compared to bispecific molecules containing scFv 1210 in the V _L -V _H format, as shown in Table 13.

Table 13. Comparison of cell binding (EC ₅₀ ) and in vitro activity in the CD137 reporter assay when scFv 1210 is in the V _H -V _L or V _L -V _H orientation

Design Design Features Affinity for human 5T4-expressing cells, EC ₅₀ (nM), measured by flow cytometry EC ₅₀ (nM) in CD137 reporter assay ALG.APV-006 V _H V _L 1618-Fc-V _H V _L 1210 15.7 0.23 ALG.APV-010 V _H V _L 1618-Fc-V _L V _H 1210 50.9 2.60

[00269] The length of the linker between the V _H and V _L domains was also assessed for scFv 1618 and 1210. Changing the length of the linker connecting the V _H and V _L with a 4-fold or 3-fold repeat of the Gly4Ser linker did not appear to result in to significant differences in activity or stability (data not shown), so 4-fold replicates were typically used unless otherwise noted.

Example 2. Optimization of scFv binding domains for CD137 1618 and 5T4

[00270] Following the initial characterization of the ADAPTIR™ bispecific construct (ALG.APV-006), it was desirable to increase 1) the binding affinities of the scFv binding domains to their respective targets; 2) biophysical stability; and 3) in vitro activity. Improvements in these parameters are expected to result in improved product costs, ease of manufacture, and reduced clinical dose sizes. To optimize binding domains, random mutagenesis phage libraries were generated for scFV 1618 and 1210 using error-prone PCR. Briefly, each scFv was used as a template in an error-prone PCR reaction using a commercially available mutagenesis kit (GeneMorph II Random Mutagenesis Kit, Agilent Technologies) according to the manufacturer's protocol. The error-prone PCR products were digested, ligated into the phagemid-scFV vector, and transformed into competent E. coli SS320/M13K07 cells to generate phage libraries. Five rounds of panning were performed for each library using biotinylated ECD 4-1BB or 5T4 as the antigen (for binding domains 1618 or 1210, respectively). For each subsequent cycle, increased panning stringency was used, decreasing the antigen concentration and increasing the washing time. After the last round of panning, the resulting phages were seeded and prepared for bulk cloning of the scFv pool into the prepared ADAPTIR ^™ expression vector. Approximately 400 individual colonies were selected, phagemids were isolated and sequenced, and purified DNA was used for high-throughput, small-scale transient transfection into 293 cells (in a culture volume of ~0.6 ml). 5-day purified supernatants were analyzed for binding by flow cytometry and SPR. The most effective variants were then subjected to a series of tests, including cell binding, in vitro activity and various stability assays. These bispecific ADAPTIR ^™ variants were generated with changes to either scFv 1210 or 1618, while maintaining the other scFv as the original sequence unchanged. This simplified the interpretation of the data and allowed the authors to evaluate the impact of these individual changes on binding, activity and stability. Based on improvements in stability, affinity, or biological activity, beneficial point mutations have been identified. These were then combined to produce an additional set of ADAPTIR DNA constructs. These constructs included several changes to scFv 1210 and/or 1618. Each construct was expressed in HEK-293 cells by transient transfection in a 250 ml culture volume and purified using protein A and SEC purification steps. Final protein purity was confirmed by analytical SEC and endotoxin levels were determined using the Endosafe PTS instrument according to the manufacturer's instructions to ensure that the in vitro activity assay results were not biased by the presence of endotoxin. The resulting protein was buffered with PBS as part of the SEC purification process, concentrated to 1 mg/mL, subjected to sterilization filtration, and stored at 4°C until next use. Protein concentration was determined by absorbance at 280 nm using the theoretical extinction coefficient.

Example 3: Evaluation of phage-derived optimized variants

[00271] The phage panning process identified several beneficial amino acid changes in scFv 1210 and 1618 compared to the parent construct ALG.APV-006. One measure of colloidal stability of a protein is to examine the amount of protein that precipitates out of solution when ammonium sulfate is added to a final concentration of approximately 1 M. Under these conditions, the original ALG.APV-006 design lost almost 89% of the protein in solution (Table 14). In comparison, the changes represented by ALG.APV-099, ALG.APV-127, ALG.APV-148 and ALG.APV-150 all show reduced protein loss under these conditions, suggesting that the individual amino acid changes made in these constructs, improved the colloidal stability of the bispecific ADAPTIR™ constructs. Another measure of protein stability is to examine the effect of applying a shear force to a sample of the protein and examine that sample for loss through precipitation or formation of soluble aggregated forms of the bispecific protein. When evaluated in a shear assay, in which a protein solution was placed in a 96-well plate and shaken at 2000 rpm for two hours, the ALG.APV-127 mutation showed a significantly reduced amount of protein loss, whereas other variants showed little or no effect aggravated loss (Table 14). Another measure of thermal stability can be determined by using differential scanning calorimetry (DSC) or differential scanning fluorimetry (DSF) to determine the temperature at the midpoint of the melting curve, otherwise known as Tm. After identifying the Tm variants 1618 and 1210, the authors noted that the ALG.APV-127 and ALG.APV-150 variants showed a three to four degree increase in the Tm of scFv 1618 compared to the parent sequence ALG.APV-006 (Table 14). An increase in Tm can generally be interpreted as an improvement in the stability of the folded protein, as it means the protein is more resistant to heat unfolding/denaturation. ALG.APV-148 increased the thermal stability of scFv 1210, as the Tm was increased by almost three degrees compared to the original 1210 sequence used in ALG.APV-006.

[00272] Various combinations of the ALG.APV-099, ALG.APV-127, ALG.APV-148 and ALG.APV-150 mutations were evaluated to understand whether the combination mutations provide additive beneficial effects on the biophysical stability of the ADAPTIR™ bispecific constructs . They were expressed and purified using the same method and assessed in the same assays as individual point mutations. The results in Table 14 show that the combination of mutations included in ALG.APV-178 and ALG.APV-179, as illustrative examples, resulted in increased stability that was generally equivalent to or better than that achieved by the individual changes.

Table 14. Biophysical Evaluation Data for Non-Optimized and Optimized ADAPTIR™ Bispecific Constructs

Design Modified domain Solubility in 1 M ammonium sulfate, % protein loss Shear stress
% protein loss Tm (°C)
scFv 1618 Tm (°C)
scFv 1210 ALG.APV-006 Original sequence 1210 and 1618 89 64 56.1 70.5 ALG.APV-099 1210 67 80 57 70.5 ALG.APV-127 1618 55 32 60.4 71.0 ALG.APV-148 1210 24 76 56.9 73.0 ALG.APV-150 1618 56 71 58.9 70.8 ALG.APV-178 1210 and 1618 18.7 24.5 61.7 72.3 ALG.APV-179 1210 and 1618 28.9 26.5 61.7 72.3

[00273] In addition to the stability assessments performed, the binding affinities of ALG.APV-006 and phage-derived variants were determined by surface plasmon resonance (SPR) using a Biacore T-200 using recombinant extracellular domains of human 5T4 and human CD137. Modifications to ALG.APV-127 and ALG.APV-150 resulted in significantly higher binding affinity to CD137 (Table 15). Similarly, ALG.APV-099 was observed to bind to a significantly higher extent than ALG.APV-006 (13 nM versus 149 nM, respectively).

Table 15. Summary of Binding and Activity Assessments for Non-Optimized and Optimized ADAPTIR™ Bispecific Constructs

Design Modified domain Affinity for Humans
CD137 by SPR method
KD (nM) Affinity for Humans
5T4 by SPR method
KD (nM) In vitro activity
EC ₅₀ (nM) ALG.APV-006 Source sequences 128 149 0.23 ALG.APV-099 1210 No data 13 0.09 ALG.APV-127 1618 58 No data 0.03 ALG.APV-148 1210 No data 198 0.05 ALG.APV-150 1618 97 No data 0.03

[00274] The binding of ALG.APV-099, ALG.APV-127, ALG.APV-148 and ALG.APV-150 variants to CD137 and 5T4 expressed on the cell surface was compared with the binding of ALG.APV-006. Despite the improved affinity for soluble CD137 or 5T4 exhibited by some of these mutations, no significant differences were observed in cell surface binding experiments (data not shown). To compare the effectiveness of phage display-derived variants in inducing target-dependent activation of CD137, variants were compared in a CD137 reporter assay.

[00275] CD137 reporter assay: Jurkat/CD137 transfectants carrying a luciferase reporter gene under the control of the NF-κB promoter (Promega) were cultured according to the manufacturer's protocols. Jurkat/NF-κB reporter cells were cultured with human 5T4-transfected CHO-K1 cells at approximately 15,000 reporter cells per 30,000 target cells in 96-well plates. Concentrations of bispecific molecules were added with final concentrations ranging from 10 nM to 0.002 nM. Cells were cultured in a total volume of 100 μl of RPMI 1640 medium supplemented with 1% fetal bovine serum, sodium pyruvate, antibiotics, and non-essential amino acids. The plates were incubated at 37°C, 5% CO2 in humidified incubators for 5-6 hours. One hundred microliters of Bio-Glow buffer (Promega) was added to each well and incubated for 5-10 minutes before fluorescence measurement. Luminescence was measured on a MicroBeta ² 2450 microplate counter (Perkin Elmer). Nonlinear regression analysis to determine EC ₅₀ values was performed using GraphPad Prism 6® graphing and statistical software.

[00276] In FIG. Figure 1 shows that all constructs showed agonistic function in the presence of 5T4(+) cells; in the absence of 5T4(+) cells, no reporter activity was observed (not shown). Constructs ALG.APV-099, ALG.APV-127, ALG.APV-148 and ALG.APV-150 showed better agonist function (lower EC ₅₀ values) compared to the parent construct ALG.APV-006. Each point on the curve represents the average of parallel wells. The Morrison format molecule ALG.APV-004 was included for comparison. The y-axis shows values in relative fluorescence units (RLU), presented as a percentage of maximum fluorescence.

[00277] Since improvements were achieved through individual point mutations represented by ALG.APV-099, ALG.APV-127, ALG.APV-148 and ALG.APV-150, these mutations were combined in various ways to determine whether whether the characteristics of individual mutations provide additive beneficial effects when incorporated into the same protein. These unique mutations were evaluated in combination to produce scFv 1618 and 1210 variants with significantly improved properties, such as ALG.APV-178, ALG.APV-179, ALG.APV-187, ALG.APV-191, ALG.APV-196 , ALG.APV-198 and ALG.APV-199. To create constructs ALG.APV-191, ALG.APV-196, ALG.APV-198 and ALG.APV-199, individual point mutations were additionally combined with the LO1, LO2 and LO11 mutations described in examples 22 and 23 of PCT Application No. PCT/ EP2017/059656. In addition, targeted phage libraries were generated after analyzing the scFv 1618 and 1210 regions to identify regions that may contribute to instability. However, panning of these libraries did not produce scFvs with improved properties (data not shown).

Example 4: Evaluation of Stabilizing Disulfide Bonds

[00278] Point mutations were made to include an additional cysteine residue in the V _H and V _L domains of both scFv 1618 and 1210. When these bispecific molecules are expressed, these cysteines react to form a disulfide bond and may contribute to increased scFv stability and confer beneficial properties for improving the production and storage of these products. Experiments have been conducted demonstrating that when the optimized bispecific molecules with a stabilizing disulfide bond are stored in scFv 1210 for one week at 4 or 25°C or when subjected to three freeze-thaw cycles from -80°C to room temperature, they retain their purity better than bispecific molecules without a disulfide bond. Exemplary data is presented in Table 16. The addition of a stabilizing disulfide bond to scFv 1618 was also evaluated. Although the disulfide bond appears to provide a stability advantage, the Biacore binding data suggests that the sample had heterogeneity resulting in atypical binding kinetics ( data not shown).

Table 16. Improved storage and freeze-thaw stability of bispecific molecules containing a stabilizing disulfide bond in scFv 1210

Design Does scFv 1210 contain a stabilizing disulfide bond? Decrease in % purity
Day 7 at 4˚C Decrease in % purity
7 day at 25˚C Change in % purity after 3 freeze-thaw cycles from
-80 ^o C to room temperature ALG.APV-178 No 2.4 1.9 4 ALG.APV-179 No 1.5 1.7 4 ALG.APV-187 Yes 0.1 0.1 1 ALG.APV-191 Yes 0.0 0.0 2 ALG.APV-196 Yes 0.0 0.1 2 ALG.APV-198 Yes 0.1 0.1 2 ALG.APV-199 Yes 0.0 0.1 1

[00279] The improvement in the stability of the bispecific molecule with the inclusion of a stabilizing disulfide bond in scFv 1210 was also clear in subsequent evaluations performed at 40°C. The two designs, ALG.APV-209 and ALG.APV-210, differ by the addition of a stabilizing disulfide bond to scFv 1210 in ALG.APV-210. After one week at this elevated temperature, ALG.APV-210 formed significantly less aggregate (Table 17).

Table 17. Improved stability data at 40°C for the bispecific molecule with a stabilizing disulfide bond in scFv 1210

Design Does scFv 1210 contain a stabilizing disulfide bond? Decrease in % purity
7 day at 40˚C ALG.APV-209 No 2.6 ALG.APV-210 Yes 0.2

[00280] While the inclusion of a disulfide bond in 1210 provided a significant advantage in terms of storage stability, other stability parameters that were evaluated did not show a significant difference between the two designs (Table 18). Both constructs performed significantly better in these assays compared to the parent bispecific molecule ALG-006 (Table 14).

Table 18. Comparison of stability data of ALG.APV-209 with ALG.APV-210

Design Does scFv 1210 contain a stabilizing disulfide bond? Modified domain Solubility in 1 M ammonium sulfate, % protein loss Shear stress
% protein loss Tm (°C) scFv 1618 Tm (°C) scFv 1210 ALG.APV-209 No 1210 and 1618 16.2 22.1 61.2 72.7 ALG.APV-210 Yes 1210 and 1618 13.1 23.1 61.5 73.2

Example 5. Assessment of binding affinity of scFv2Fc bispecific proteins to human 5T4 and human CD137

Methods

[00281] Expression and purification of recombinant extracellular domains (ECD) of human 5T4 and human CD137: Using standard molecular biology techniques and starting from a vector encoding the full-length protein sequence of 5T4 and CD137, nucleotide primers were designed to amplify the extracellular regions of human 5T4 and human CD137 . An additional peptide sequence was added to the c-terminus of the ECD at a position where the native protein is expected to enter the membrane. This peptide contained recognition sequences that can be used for affinity purification purposes. Expression vectors were transiently transfected into HEK-293 cells. Recombinant ECD was purified from conditioned media using immobilized metal ion metal affinity chromatography (IMAC) and size exclusion chromatography. Protein purity was confirmed by analytical SEC.

[00282] Surface plasmon resonance (SPR) affinity assays of bispecific proteins for recombinant ectodomain CD137 and 5T4: SPR binding affinity studies of bispecific proteins for 5T4 x CD137 with recombinant monomeric ectodomain (ECD) 5T4 and CD137 were performed at 25°C in HBS buffer -EP ⁺ in Biacore T200 system. Goat anti-human IgG F(ab')2 fragment antibody (Jackson ImmunoResearch) at a concentration of 20 μg/ml in 10 mM sodium acetate (pH 4.5) was immobilized at a density of 2000 response units (RU) in the flow cell of the CM5 research sensor chip. (GE) via standard immobilization at the amino group. Each bispecific variant at a concentration of 200 nM in HBS-EP+ buffer was captured in a flow cell with immobilized anti-human IgG antibody at a flow rate of 50 μl/min for 60 seconds to achieve a response of ~500 RU, while the surface of one flow cell was left unchanged at quality of control. After a 30-second stabilization period, four different concentrations of each ECD (0, 20, 60, and 180 nM) were injected at 50 μL/min for either 120 seconds followed by a dissociation period of 240 seconds (for 5T4) or 90 seconds followed by a dissociation period dissociation 180 seconds (for 4-1BB). Regeneration was carried out by double injections of 10 mM glycine (pH 1.5) at a flow rate of 50 μl/min for 30, followed by stabilization with HBS-EP+ buffer for 1 min.

[00283] Sensorgrams obtained from SPR kinetics measurements were analyzed by double subtraction. The signal from the control flow cell was subtracted from the analyte binding response obtained from flow cells with immobilized or entrapped ligands. Buffer control responses were then averaged across multiple injections. The averaged buffer control responses were then subtracted from the analyte binding responses, and the final double-control data were analyzed using Biacore T200 Evaluation software (2.0, GE), performing a global fit to the data to obtain kinetic parameters. All sensorgrams were fitted using a simple one-to-one binding model.

results

[00284] Table 19 shows the binding parameters determined by SPR for the ADAPTIR™ bispecific constructs binding to CD137 and 5T4. It should be noted that performing affinity measurements in this format by capturing the bispecific molecule on a chip and introducing recombinant monomeric CD137 or 5T4 ECD should result in true affinity values that are not confounded by avidity effects. As shown in Table 19, through careful screening of randomized phage libraries, binding domain variants were identified that resulted in significant improvements in scFv binding affinity to CD137 and/or 5T4. Compared to the non-optimized parent design ALG.APV-006, the optimized ADAPTIR™ bispecific designs ALG.APV-178, ALG.APV-179, ALG.APV-187, ALG.APV-209 and ALG.APV-210 achieved a large reduction in value KD to human CD137. Similarly, ALG.APV-179, ALG.APV-187, ALG.APV-209 and ALG.APV-210 showed improved affinity values for human 5T4 compared to non-optimized ALG.APV-006.

Table 19. Binding affinity constants ( _KD ), dissociation and association for bispecific molecules binding to recombinant human CD137 or human 5T4

Affinity for human CD137
SPR method Affinity for human 5T4
SPR method Design KD (nM) k _a (1/Ms) k _d (1/s) KD (nM) k _a (1/Ms) k _d (1/s) ALG.APV-006 210 2.8E+05 5.9E-02 109 2.4E+04 2.6E-03 ALG.APV-178 70 3.9E+05 2.7E-02 106 2.3E+04 2.5E-03 ALG.APV-179 71 3.9E+05 2.8E-02 29 2.2E+04 6.2E-04 ALG.APV-187 73 3.7E+05 2.7E-02 35 2.1E+04 7.2E-04 ALG.APV-209 59 5.6E+05 3.3E-02 66 1.4E+04 9.4E-04 ALG.APV-210 59 5.5E+05 3.3E-02 79 1.5E+04 1.2E-03

Example 6. Evaluation of the thermal stability of scFv-Fc-scFv bispecific proteins to human 5T4 and human CD137

[00285] Differential scanning calorimetry (DSC) and differential scanning fluorimetry (DSF) are tools commonly used to measure the structural stability of recombinant proteins. By measuring the energy required to raise the temperature of a protein sample, the average melting temperature (Tm) of individual protein domains can be determined. It is generally accepted that protein domains with higher Tm values are considered more stable. DSF analysis was performed using a set of optimized ADAPTIR ^™ bispecific proteins (ALG.APV-178, ALG.APV-179 and ALG.APV-187) and compared with a non-optimized bispecific version of ADAPTIR ^™ (ALG.APV-006) (Table 20). Significant increases in Tm 1618 to CD137 and 1210 to 5T4 were observed after inclusion of beneficial mutations that were identified using the random mutagenesis phage display approach described above.

Table 20. Average melting temperature, Tm, of optimized ADAPTIR™ bispecific constructs compared to non-optimized constructs

Optimized? Design Tm, scFv to CD137, °C Tm, scFv to 5T4, °C No ALG.APV-006 56.5 70.2 Yes ALG.APV-178 60.7 73.0 Yes ALG.APV-179 61.1 73.0 Yes ALG.APV-187 61.1 73.5

Example 7. Binding of scFv-Fc-scFv bispecific protein molecules to human and non-human primate CD137(+) cell lines.

[00286] To confirm that cell surface CD137 binding was not lost following conversion of variable domains to scFvs or introduction of mutations, flow cytometry was used to quantify the binding of engineered CD137 x 5T4 ADAPTIR™ bispecific molecules to human CD137-expressing cell lines or cynomolgus macaque.

[00287] Binding studies on CD137-expressing CHO-K1 cells were performed using standard flow cytometry-based staining procedures. CHO-K1 cells (obtained from Alligator) were transfected with human or cynomolgus CD137. In a typical experiment, approximately 100,000 cells per well in 96-well plates were labeled with 100 nM to 0.05 nM binding molecule in 50 μl saline buffer with 2% BSA and 2 mM EDTA for 30 min on ice, followed by washes and by incubation with PE-labeled secondary antibody, goat anti-human IgG Fcγ antibody (Jackson Laboratory), for 20 minutes on ice. The signal from bound molecules was detected using an LSR-IITM or a Symphony A3 flow cytometer (BD Biosciences) and analyzed using FlowJo flow cytometry data analysis software. The median fluorescence intensity (median MFI) of bound molecules on live cells was determined after excluding cell doublets. Nonlinear regression analysis to determine EC ₅₀ values was performed using GraphPad Prism 6® graphing and statistical software.

[00288] In FIG. Figure 2 shows the binding curves of seven ADAPTIR™ molecules (ALG.APV-006, ALG.APV-178, ALG.APV-179, ALG.APV-187, ALG.APV-191, ALG.APV-196 and ALG.APV-198 ) with the human CHO-K1/CD137 cell line. All molecules showed similar levels of binding (EC ₅₀ values) and saturation. In FIG. Figure 3 shows the binding curves of the same seven constructs to the cynomolgus cell line CHO-K1/CD137. Similar levels of binding and saturation were observed for all seven constructs for the cynomolgus target. In FIG. 12 shows the binding curves of six ADAPTIR™ molecules (ALG.APV-178, ALG.APV-179, ALG.APV-187, ALG.APV-208, ALG.APV-209 and ALG.APV-210) and control in Morrison format ALG.APV-004 with human CHO-K1/CD137 cell line. All molecules bound to similar EC ₅₀ values of 0.3 to 0.6 nM and similar saturation levels.

Example 8. Binding of scFv-Fc-scFv bispecific protein molecules to human and non-human primate 5T4(+) cell lines.

[00289] To compare 5T4 binding on the surface of cancer cells following changes in scFv domains, flow cytometry was used to quantify the binding of engineered CD137 x 5T4 bispecific binding molecules ADAPTIR ^™ to 5T4 expressing cell lines.

[00290] Binding of CD137 x 5T4 bispecific molecules to 5T4(+) cell lines: binding characteristics of ADAPTIR™ bispecific molecules (ALG.APV-178, ALG.APV-179, ALG.APV-187, ALG.APV-196, ALG) were compared .APV-208, ALG.APV-209 and ALG.APV-210). Binding studies were performed using standard flow cytometry-based staining procedures on two 5T4-expressing cell lines: CHO-K1 cells (derived from Alligator) transduced with human or cynomolgus 5T4, and human ovarian adenocarcinoma SKOV-3 cells expressing (human) 5T4 . All labeling and washings were performed in 96-well U-bottom plates in saline buffer with 0.1% BSA and 2 mM EDTA. Cells were seeded at a density of approximately 100,000 cells per well and incubated with test molecules at concentrations ranging from 100 nM to 0.05 nM in a volume of 50 μl/well for 30 minutes on ice. Cells were washed three times and then incubated for an additional 30 min on ice with fluorescently labeled secondary polyclonal goat anti-human IgG F(ab') ² antibody (Jackson ImmunoResearch Laboratories). Cells were then washed twice and samples were injected into a BD LSRII or Symphony A3 flow cytometer. Sample files were analyzed using FlowJo software; the mean fluorescence intensity (mean MFI) or median fluorescence intensity (median MFI) for the living cell population in each well was calculated after gating of live cells (from forward and side scatter signals). Nonlinear regression analysis to determine EC ₅₀ values was performed using GraphPad Prism 6® graphing and statistical software.

[00291] In FIG. Figure 4 shows the binding curves of seven ADAPTIR™ molecules (ALG.APV-006, ALG.APV-178, ALG.APV-179, ALG.APV-187, ALG.APV-191, ALG.APV-196 and ALG.APV-198 ) with the human CHO-K1/5T4 cell line. The ALG.APV-006 construct was associated with an EC ₅₀ value > 10 nM, whereas the remaining ADAPTIR™ constructs (ALG.APV-178, ALG.APV-179, ALG.APV-187, ALG.APV-191, ALG.APV- 196 and ALG.APV-198) were associated with EC ₅₀ values of 3 to 10 nM. Differences in EC ₅₀ values between constructs were more pronounced when binding assays were performed on SKOV-3 cells. SKOV-3 cells express human 5T4 at levels approximately 10-fold lower than human CHO-K1/5T4 transfectants (FIG. 5). In FIG. Figure 6 shows the binding curves of the ALG.APV-006, ALG.APV-178, ALG.APV-179, ALG.APV-187, ALG.APV-196 and ALG.APV-198 constructs obtained on SKOV-3 cells. The ALG.APV-006, ALG.APV-078, ALG.APV-179, and ALG.APV-187 constructs bound with slightly lower affinity (higher EC ₅₀ values) and higher maximum binding levels than the ALG.APV- constructs. 196 and ALG.APV-198. In FIG. Figure 7 shows the binding curves of ALG.APV-006, ALG.APV-178, ALG.APV-179, ALG.APV-187, ALG.APV-191, ALG.APV-196 and ALG.APV-198 on CHO-K1 transfectants /5T4 cynomolgus macaque. Similar to the binding profile on human 5T4(+) cells of designs ALG.APV-006, ALG.APV-178, ALG.APV-179, ALG.APV-187, ALG.APV-191, ALG.APV-196 and ALG.APV -198 showed lower affinity and higher maximum binding levels than the ALG.APV-196 and ALG.APV-198 constructs.

[00292] In FIG. 13 shows the binding curves of six ADAPTIR™ molecules (ALG.APV-178, ALG.APV-179, ALG.APV-187, ALG.APV-208, ALG.APV-209 and ALG.APV-210) and control in Morrison format ALG.APV-004 with mouse CT26 cells (ATCC) expressing human 5T4. These cells express human 5T4 at levels comparable to those observed for SKOV-3 cells (FIG. 5). All constructs bound to EC ₅₀ values between 1 and 3 nM. The constructs ALG.APV-179, ALG.APV-187, ALG.APV-209, and ALG.APV-210 showed higher levels of maximal binding than ALG.APV-178 and ALG.APV-208. The control molecule in Morrison format ALG.APV-004 showed the lowest levels of maximum binding. In FIG. 14 shows the binding curves of ALG.APV-004, ALG.APV-178, ALG.APV-179, ALG.APV-187, ALG.APV-208, ALG.APV-209 and ALG.APV-210 on CHO-K1 transfectants /5T4 cynomolgus macaque. All molecules bound with similar EC ₅₀ values of 6 to 10 nM and similar saturation levels. In FIG. 15 shows the binding curves of ALG.APV-004, ALG.APV-178, ALG.APV-179, ALG.APV-187, ALG.APV-208, ALG.APV-209 and ALG.APV-210 with a human cell line, expressing neither CD137 nor 5T4 (MOLM13, ATCC cells). Neither molecule showed binding at any concentration.

Example 9. Binding of bispecific antibody to 5T4-CD137 to 5T4-expressing cell lines.

[00293] Experiments were performed to determine the binding of ALG.APV-210 to human 5T4 expressed on cells at different receptor densities. Both a human tumor cell line with endogenous levels of 5T4 and cells transfected to produce 5T4 were assessed for targeted binding of ALG.APV-210.

Materials and methods

[00294] Biotinylation: To detect the binding of ALG.APV-210 to 5T4-expressing cell lines, ALG.APV-210 was first biotinylated. The quality of the biotinylated protein and the effect on binding to its targets were assessed using ELISA.

[00295] FACS staining and analysis of 5T4-expressing cell lines: Prior to FACS staining, all cell lines were characterized for their 5T4 expression levels using a receptor density kit (Quantum Simply Cellular, anti-human IgG, Bangs Laboratories, Inc). Information about the cell lines used and their respective 5T4 expression densities is presented in Table 21. Attached cell lines were cultured according to their supplier's instructions and collected from the flasks in which they were cultured using trypsin/EDTA, counted in R10 medium in a Bürker counting chamber and diluted with FACS buffer (PBS + 0.5% BSA, 0.05% NaN3) to a concentration of 4x 10 ⁶ cells/ml. 50 µl (0.20 x 10 ⁶ ) of cell solution was added to a 96-well FACS staining plate (Falcon #351190). Cells were incubated for 20 min at 4°C with 50 μl of biotinylated ALG.APV-210 (2 times the final concentration). Serial dilutions of antibodies were added, starting with a titer of 20 μg/ml, with 12-step dilutions of 2 times to 0.010 μg/ml. After washing twice with FACS buffer, the cells were incubated for 30 min at 4°C with 100 μl of secondary antibody (streptavidin-PE, BD no. 554061, diluted 1/500). After two washing steps with FACS buffer, cells were resuspended in 200 μl of cell fixation reagent (BD CellFIX 10x BD, no. 340181, diluted in MQ H2O). Cells were analyzed for ALG.APV-210 binding using flow cytometry (FACSVerse) by single cell gating. MFI values (subtracted secondary antibody only) from 1 of 2 independent experiments, as well as normalized data and EC ₅₀ values of pooled data from 2 experiments were determined and plotted using GraphPad Prism 7, non-linear regression of log(agonist) on normalized response - variable slope.

Table 21. 5T4 Expressing Cell Lines and Receptor Densities/Cell

Cells Type of cancer Density 5T4/cell EC ₅₀ (nM) 95%CI NST-116 Human colon carcinoma 62,000 16 15-17 HT-29 Human colon carcinoma 30,000 12 11-14 JAR Human choriocarcinomas 69,000 9 5-17 J.E.G. Human choriocarcinomas 66,000 8 5-12 SKOV-3 Human ovarian cancer 60,000 15 12-18 BxPC-3 Pancreas cancer 150,000 20 18-22 B16-hu5T4 Transfected, mouse melanoma 320,000 14 9-19 CHO-hu5T4 Transfected 4,100,000 23 18-28 CT26-hu5T4 ^high Transfected, mouse colon carcinoma 1,000,000 20 16-27 CT26-hu5T4 ^medium Transfected, mouse colon carcinoma 200,000 12 11-13 CT26-hu5T4 ^low Transfected, mouse colon carcinoma 50,000 eleven 8-15

Results and conclusions

[0029] Using ELISA, the binding of biotinylated ALG.APV-210 to 5T4 was found to be similar to that of non-biotinylated ALG.APV-210 (data not shown).

[00297] The binding (EC ₅₀ ) of biotinylated ALG.APV-210 to 5T4 when expressed on cells, either at endogenous levels in human tumor cells or on cells transfected to express 5T4, was assessed. As shown in FIG. 39 by MFI, ALG.APV-210 binds in a dose-dependent manner to both human tumor cells expressing endogenous levels of 5T4 and to transfected cell lines.

[00298] Table 21 above shows the EC ₅₀ of binding of ALG.APV-210 to a 5T4-positive human tumor cell line and 5T4-transfected cells determined from normalized MFI values (minus background values) from 2 combined experiments plotted dose-response shown in FIG. 40. Average EC ₅₀ values were 8-23 nM for different 5T4-positive cell lines. Thus, ALG.APV-210 binds to 5T4-expressing cells with an EC ₅₀ of 8-23 nM.

Example 10 Agonist function of scFv-Fc-scFv bispecific proteins in reporter assays

[00299] To compare the effectiveness of different CD137 bispecific binding molecules in inducing target-dependent activation of CD137, seven different CD137 x 5T4 bispecific molecules were compared in a CD137 reporter assay as described in Example 3.

CD137 reporter assay

[00300] Jurkat/CD137 transfectants carrying a luciferase reporter gene under the control of the NF-κB promoter (Promega) were cultured according to the manufacturer's protocols. Jurkat/NF-κB reporter cells were cultured with human primary ductal breast carcinoma HCC1143 (ATCC) cells expressing human 5T4 at approximately 15,000 reporter cells per 30,000 target cells in 96-well plates. Concentrations of bispecific molecules were added with final concentrations ranging from 10 nM to 0.002 nM. Cells were cultured in a total volume of 100 μl of RPMI 1640 medium supplemented with 10% fetal bovine serum, sodium pyruvate, antibiotics, and non-essential amino acids. The plates were incubated at 37°C, 5% CO ₂ in humidified incubators for 5-6 hours. One hundred microliters of Bio-Glow buffer (Promega) was added to each well and incubated for 5-10 minutes before fluorescence measurement. Luminescence was measured on a MicroBeta ² 2450 microplate counter (Perkin Elmer). Nonlinear regression analysis to determine EC ₅₀ values was performed using GraphPad Prism 6® graphing and statistical software.

results

[00301] In FIG. 8 all constructs show agonistic functional activity in the presence of 5T4(+) cells; in the absence of 5T4(+) cells, no reporter activity was observed (data not shown). The ALG.APV-178, ALG.APV-179, ALG.APV-187, ALG.APV-191, ALG.APV-196 and ALG.APV-198 constructs showed better agonistic functional activity (up to 6-fold lower values EC ₅₀ ) compared to the non-optimized ADAPTIR ^™ design, ALG.APV-006, and the Morrison format control design ALG.APV-004. Each point on the curve represents the average of parallel wells. The y-axis shows values in relative fluorescence units (RLU). In FIG. 16 shows the activity of bispecific constructs in Jurkat/NF-κB reporter cells after 5 hours of incubation in the presence of 5T4(+) cells (HCC1143) or 5T4(-) cells (MOLM13). The ALG.APV-178, ALG.APV-179, ALG.APV-187, ALG.APV-208, ALG.APV-209 and ALG.APV-210 constructs showed better agonistic functional activity (up to 10-fold lower values EC ₅₀ ) compared to the Morrison format control design ALG.APV-004. No reporter activity was observed in the presence of 5T4(-) cells (MOLM13). Each point on the curve represents the average of parallel wells. The y-axis shows values in relative fluorescence units (RLU).

Example 11 Agonist function of scFv-Fc-scFv bispecific proteins in primary T cell assays

[00302] The agonist function of various CD137 x 5T4 bispecific molecules in inducing target-dependent activation of CD137 was tested in peripheral blood mononuclear cell (PBMC) cultures. To test the function of CD137 bispecific constructs, primary PBMCs were stimulated with anti-CD3 antibodies to increase the expression of CD137, which is not expressed on resting T cells. Co-stimulation of primary T cells by TCR and CD137 enhances T cell secretion of the cytokine interferon-gamma (IFN-γ).

Stimulation of PBMCs via CD137:

[00303] Peripheral blood mononuclear cells (PBMCs) were isolated from human blood using standard Ficoll gradients. The isolated cells were washed in saline buffer. PBMCs were cultured with human CHO-K1/5T4 cells at a rate of approximately 120,000 PBMCs per 30,000 5T4(+) cells in 96-well plates. Anti-CD3 antibody OKT3 (eBioscience) was added to all wells at a concentration of 0.1 μg/ml. Cells were cultured in a total volume of 200 μl of RPMI 1640 medium supplemented with 1% fetal bovine serum, sodium pyruvate, antibiotics, and non-essential amino acids. The plates were incubated at 37°C, 5% CO ₂ in humidified incubators for 72 hours. After 72 hours, one hundred microliters of medium was collected. IFN-γ levels in the supernatant were measured using Milliplex® kits (Millipore) according to the manufacturer's instructions. Data were collected on a Bio-Plex Reader 200 system (Bio-Rad). Nonlinear regression analysis to determine EC ₅₀ values was performed using GraphPad Prism 6® graphing and statistical software.

[00304] In FIG. Figure 9 shows IFN-γ levels induced by ALG.APV-006, ALG.APV-178, ALG.APV-179, ALG.APV-187, ALG.APV-196, ALG.APV-198, and a control construct in Morrison format ALG.APV-004. All constructs induced higher levels of IFN-γ than anti-CD3 alone in a dose-dependent manner. Each point on the curve represents the average of parallel wells. In FIG. 17 shows the levels of IFN-γ induced in primary PBMC cultures after 72 hours by ALG.APV-178, ALG.APV-179, ALG.APV-187, ALG.APV-208, ALG.APV-209 and ALG.APV- 210, and a control construct in Morrison format, ALG.APV-004, in the presence of human CHO-K1/5T4 cells. All constructs induced higher levels of IFN-γ than anti-CD3 alone in a dose-dependent manner. Each point on the curve represents the average of parallel wells.

[00305] The functional activity of the 5T4 x CD137 bispecific constructs was also assessed in a CD8+ T cell assay in which the cells were cultured in microtiter plates coated with the 5T4-Fc construct and anti-CD3 antibody.

Stimulation of purified T cells with 5T4:

[00306] PBMCs were isolated by density gradient centrifugation using Ficoll-Paque (ρ 1.077 g/ml) (GE Healthcare No. 17-1440-02) from leukocyte concentrates obtained from healthy donors (Clinical Immunology and Transfusion Medicine, Lund, Sweden). CD8+ T cells were enriched through negative selection using a CD8+ T cell isolation kit (Miltenyi 130-096-495). Plates were coated overnight at 4°C with 3 μg/ml α-CD3 clone OKT3 (Affymetrix eBioscience no. 16-0037-85), washed and coated with 5 μg/ml 5T4-Fc for 2 h at 37°C. After coating with 5T4-Fc, plates were washed and blocked for a minimum of 30 minutes with RPMI (Gibco #61870010) containing 10% FCS (heat inactivated, Gibco #10270-106, lot 41Q9248K) and 10 mM Hepes (Gibco #15630056). Bispecific constructs were diluted in RPMI containing 10% FCS and 10 mM Hepes and added to the plates 30 minutes before adding CD8+ T cells (0.07 × 10 ⁶ cells/well). The assay plates were incubated for 72 h at 37°C, after which the culture supernatants were collected. IFN-γ levels in the supernatants were measured by ELISA (BD OptiEIA no. 555142). The experiment was repeated twice with a total of 3 donors. The results of the first experiment are shown in FIG. 11A. Results for control samples in which cells and bispecific constructs were cultured in plates that were not coated with 5T4-Fc are shown in FIG. 11B.

[00307] In FIG. 11A shows that bispecific constructs for 5T4 x CD137 in the ADAPTIR ^™ format (ALG.APV-178, ALG.APV-179, ALG.APV-196 and ALG.APV-198) have increased functional activity compared to bispecific constructs in the format Morrison (ALG.APV-004), as evidenced by increased levels of IFN-γ present in culture supernatants in the presence of 5T4. In fact, the ADAPTIR ^™ format constructs induced IFN-γ levels that were 2-4.5 times higher than those induced by the Morrison format constructs (FIG. 11A).

Example 12. Antigen-dependent localization of bispecific antibodies in 5T4-positive tumors in vivo

[00308] Antigen-dependent localization of bispecific constructs targeting 5T4 and 4-1BB to 5T4-positive B16-5T4 tumors was assessed in wild-type C57BL/6 mice.

Materials and methods

[00309] Mice: 8-week-old female C57BL/6 mice from Janvier, France were used for experiments. All experiments were performed with the approval of the Malmö/Lund ethics committee.

[00310] Bispecific constructs: Two optimized 5T4 and CD137-targeting bispecific constructs were used in the ADAPTIR ^™ format: ALG.APV-209 and ALG.APV-210. ALG.APV-004, a bispecific construct in Morrison format, served as a positive control. Vehicle administration served as a negative control.

[00311] Cells: Wild type B16.F10 cells (B16) were obtained from ATCC and cultured according to their guidelines. B16 cells expressing human 5T4 (hereinafter referred to as B16-5T4) were obtained from Professor Peter Stern and grown in G418 selective medium at a concentration of 1.2 mg/ml.

[00312] Methods: For antigen-dependent localization of bispecific constructs, a twin B16 melanoma cell tumor model was used in which each mouse received one 5T4-negative and one 5T4-positive tumor on each side of the hind flank/back. Tumor cell lines in logarithmic growth phase were injected subcutaneously (1x10 ⁵ cells in 100 μl) on day 0. The construct (100 μg) was administered intraperitoneally on days 6 and 13, and mice were sacrificed on day 14 (24 hours after the last administration).

[00313] FACS Analysis: Tumors for flow cytometry analysis were mechanically and enzymatically digested using Liberase TL and DNase I (Roche) and passed through a 70 μm cell sieve. The resulting single cell suspension was stained for FACS analysis. Briefly, nonspecific binding of the constructs was blocked using blocking mouse IgG or Fc. Dead cells were excluded using Fixable Viability Stain 450 (Molecular probes) according to the manufacturer's instructions. Binding of two ADAPTIR ^™ bispecific constructs or control constructs to tumor cells was analyzed using a secondary antibody: goat anti-human Fc-PE (Jackson Immunoresearch). Alternatively, detection of bispecific constructs was performed using biotinylated 4-1BB antigen followed by streptavidin-PE/PerCP-Cy5.5. Samples were analyzed on BD FACSVerse and data analyzed using FlowJo software. Statistical analysis was performed using the nonparametric 2-tailed Mann-Whitney T test and GraphPad prism software.

[00314] Immunohistochemistry: Tumors for immunohistochemistry were snap frozen in isopropanol on dry ice. Frozen tissue sections (8 mm) were stained for 5T4 expression using rabbit anti-human 5T4 antibody (Abcam) or rabbit anti-human Fc antibody (Jackson Immunoresearch), followed by anti-rabbit Brightvision-HRP (Immunologic) and DAPI. Immunohistochemical staining was graded as follows: negative (0), weak staining (1+), moderate staining (2+), or strong staining (3+).

results

[00315] Both bispecific constructs in ADAPTIR ^TM format, ALG.APV-209 and ALG.APV-210, and the positive control, ALG.APV-004, localized to 5T4-expressing B16 tumors, but not to 5T4-negative tumors (B16 .F10). This was demonstrated by both flow cytometry (FIG. 18A and 18B) and immunohistochemistry (FIG. 19A and 19B).

[00316] Both the bispecific constructs (ALG.APV-209, ALG.APV-210) and the positive control (ALG.APV-004) demonstrated statistically significant 5T4-dependent localization to antigen-positive tumors compared to the negative control vehicle, according to flow cytometry data (FIG. 18A). Binding to 5T4-expressing tumors could also be detected using biotinylated 4-1BB detection (FIG. 18B), confirming that the bispecific molecules were intact.

[00317] Localization was further demonstrated by immunohistochemistry (FIGS. 19A and 19B and Table 22). Bispecific antibodies bound highly to 5T4-positive tumors (B16-5T4, FIG. 19A) but not to 5T4-negative tumors (B16.F10, FIG. 19B).

Table 22. In vivo localization of bispecific constructs and vehicle controls

Treatment 5T4-negative tumors 5T4-positive tumors ALG.APV-209 - +++ ALG.APV-210 - +++ ALG.APV-004 - +++ Carrier - -

Example 13 In vitro activity of bispecific antibody to 5T4-CD137 in an IFN-γ release assay using human CD8 T cells and 5T4-CT26 tumor cells

[00318] The functional activity of the anti-5T4-CD137 bispecific antibody ALG.APV-210 was assessed in a CD8 T cell assay in which CD8 T cells stimulated with bead-coated anti-CD3 antibodies were cocultured in plates with CT26 tumor cells. expressing different levels of 5T4.

[00319] CT26 tumor cells, a mouse colon carcinoma cell line (ATCC® CRL-2638), were pretransfected to express human 5T4 and then sorted into single cells using flow cytometry to generate single cell clones expressing either high or low human 5T4 levels (see Table 23 for receptor density). 5T4-CT26 tumor cells or untransfected wild-type cells (CT26 wild-type) were irradiated with UV radiation using a UV-B cross-linker (AnalytikJena, irradiation source: 254 nm). After irradiation of the plate, cells were washed once in medium and then resuspended at a concentration of 4 x 10 ⁶ cells/ml in new R10 growth medium (RPMI, Gibco no. 61870010, containing 10% heat-inactivated FBS, Hyclone no. SV30160, lot RB35944, and 10 mM Hepes, Gibco No. 15630056). 50 μl (2 x 10 ⁵ cells/well) of UV-irradiated CT26-h5T4 ^high , CT26-h5T4 ^low , or CT26 wild-type cells were added to tissue culture-treated assay plates (Eppendorf no. 0030 730.119) and incubated at 37°C C overnight.

Table 23. 5T4 receptor density on human 5T4 transfected and single cell sorted CT26 cells

Cell clone CT26 5T4 receptor density/cell CT26-h5T4 ^high 1 x 10 ⁶ ± 2 x 10 ⁵ CT26-h5T4 ^low 5 x 10 ⁴ ± 1 x 10 ⁴ CT26 wild type 0

[00320] The day after UV irradiation of CT26 cells, human PBMCs were isolated by density gradient centrifugation using Ficoll-Paque (GE Healthcare No. 17-1440-02) from leukocyte concentrates obtained from healthy donors (Clinical Immunology and Transfusion Medicine, Lund, Sweden). CD8+ cells were enriched through negative selection using a CD8+ T cell isolation kit (Miltenyi 130-096-495).

[00321] Serial dilutions of bispecific antibody ALG.APV-210 were prepared in R10 medium and 50 μl was added to each well of assay plates and incubated at 37°C for 30 minutes before adding 50 μl of αCD3 beads (4 x 10 ⁸ / ml of anti-CD3 antibody clone: OKT-3, Affymetrix eBioscience No. 16-0037-85, applied to beads, diluted to 2×10 ⁶ /ml) with a CD8+ T-cell to bead ratio of 1:1, followed by the addition of 50 µl of effector cells (enriched CD8+ cells, 2 × 10 ⁶ /ml or 1 × 10 ⁵ /well). Assay plates were incubated for 72 h at 37°C and the culture supernatant was collected. IFN-γ levels in the supernatants were measured by ELISA (BD OptiEIA no. 555142). EC ₅₀ was determined using a non-linear regression of log (agonist) on the normalized response (variable slope) using GraphPad Prism 7. The value of TOP (maximum functional effect) was determined by calculating the average of the two highest values of IFN-γ production in the culture supernatant. The experiment was performed 5 times, including a total of 12 donors.

[00322] The study established the functional activity of the CD137-5T4 bispecific antibody ALG.APV-210 using human CD8 T cells (effector cells) co-cultured in plates with CT26 cells expressing varying levels of 5T4 (to cross-link CD137 by binding to 5T4 ). As shown in FIG. 20, the anti-CD137-5T4 bispecific antibody ALG.APV-210 induces potent T cell activation, as measured by IFN-γ release, in a dose-dependent manner in the presence of CT26 cells expressing either high (CT26-h5T4 ^high ) or low (CT26-h5T4 ^low ) levels 5T4. The release of IFN-g is presented in FIG. 20 in the form of normalized ALG.APV-210 data; average values for 12 donors from 5 experiments combined are shown.

[00323] Table 24 presents the EC ₅₀ for ALG.APV-210 in a CD8 T cell assay in the presence of CT26 cells expressing human 5T4. Mean log (agonist) values as a function of mean normalized response values (variable slope) and 95% CI for 12 donors from 5 experiments pooled are presented in this table. As shown in FIG. 21A and 21B, the maximal functional activity of ALG.APV-210 was dependent on the level of 5T4 expression on CT26 cells, as evidenced by slightly lower secretion of maximal IFN-γ release in co-culture with CT26-h5T4 cells ^{is low} compared to co-culture with CT26 cells -h5T4 ^high (maximum activity of CT26-h5T4 ^low was 82% of CT26-h5T4 ^high ). In the absence of 5T4-induced cross-linking using wild-type CT26 cells, T cell activity is absent or very low (7% of maximal CT26-h5T4 ^high activity). The maximum release of IFN-γ is presented in FIG. 21 as absolute values (ng/ml) (FIG. 21A) or normalized values relative to CT26-h5T4 ^high (FIG. 21B). Shown are means and SEM values for 12 donors from 5 experiments combined.

Table 24. Binding (EC ₅₀ ) of ALG.APV-210 in the presence of human 5T4+ CT26 cells

ALG.APV-210-h5T4 ^high ALG.APV-210-h5T4 ^low EC ₅₀ (nM) 0.41 0.24 95% CI From 0.36 to 0.46 From 0.20 to 0.28

Example 14 Binding of the bispecific antibody to 5T4-CD137 to CD137 on primary human and cynomolgus CD3-stimulated CD8 T cells.

[00324] A study was conducted to compare the relative binding of ALG.APV-210 to human and cynomolgus CD137 expressed on activated primary CD8 T cells (gated from CD3-stimulated PBMCs).

Materials and methods

[00325] To detect binding of ALG.APV-210 to primary human and cynomolgus cells in FACS, ALG.APV-210 was first biotinylated. The quality of the biotinylated protein was assessed by HPLC, and the effect on binding to its targets was assessed by ELISA. ANC017, an ADAPTIR-formatted antibody with the same framework as ALG.APV-210 (germline CDR), was used as a negative control and was also biotinylated.

[00326] Isolation and stimulation of human PBMCs: Human PBMCs were isolated by density gradient centrifugation using Ficoll-Paque (GE Healthcare No. 17-1440-02) from leukocyte concentrates obtained from 3 healthy human donors (Clinical Immunology and Transfusion Medicine, Lund, Sweden). Non-tissue culture treated 96-well plates (Nunc #268200) were precoated overnight at 4 ºC with 10 μg/ml αCD3 clone OKT3 (Affymetrix eBioscience #16-0037-85). The next day, the plates were washed with PBS and human PBMCs diluted in R10 (RPMI, Gibco #61870010, containing 10% heat-inactivated FBS, Hyclone #SV30160, lot RB35944, and 10 mM Hepes, Gibco #15630056) were added at a concentration of 0.2 x 10 ⁶ cells/well, and incubated at 37°C in wells with or without CD3 stimulation for 48 hours.

[00327] Isolation and stimulation of cynomolgus PBMCs: Cynomolgus PBMCs were isolated from 20 ml of cynomolgus blood from 3 different donors obtained from Silabe, France, by density gradient centrifugation using Lympholyte-mammal (Cedarlane labs) according to the manufacturer's instructions . 96-well plates not treated with tissue culture were precoated overnight at 4°C with 3 μg/ml anti-simian CD3 antibody, clone FN-18 (Invitrogen no. APS0301). Cynomolgus PBMCs were then stimulated in the same manner as described above for human PBMCs.

[00328] FACS staining and CD8 T cell analysis: After incubation for 48 hours with or without CD3, human and cynomolgus PBMCs were collected, pooled, counted, and diluted in FACS buffer (PBS + 0.5% BSA, 0.05 % NaN ₃ ) to a concentration of 5×10 ⁶ cells/ml. 50 μl (0.25 x 10 ⁶ cells) of cell solution was added to a 96-well FACS staining plate (Falcon #351190). After 10 min of Fc blocking at room temperature using Beriglobin (hIgG, 200 μg/ml), cells were washed in FACS buffer and then incubated for 1 hour at 4°C with 100 μl of biotinylated ALG.APV-210 or negative control. Serial dilutions of antibodies were added, starting either from a titer of 5 μg/ml with a 12-step dilution of 3 times to 0.0003 μg/ml (exp. 1), or from a titer of 1 μg/ml with a 12-step dilution of 2 times to 0.0005 μg/ml (exp. 2). After washing twice with FACS buffer, cells were incubated for 30 min at 4°C with 50 μl of secondary antibody (streptavidin-APC, BD no. 554067) and 50 μl of fluorescently conjugated antibodies against various markers on the surface of T cells (CD4-FITC no. 550628 , CD3-PECy7 no. 557749 and CD8-APC-H7 no. 5601797, BD). After washing in PBS, cells were stained for 15 min at 4°C with 50 μl of cell viability fixative dye BV510 (to separate nonviable cells), washed again and then resuspended in 130 μl of cell fixation reagent (BD CellFIX 10x BD, no. 340181, diluted in MQ H2O). Cells were analyzed for ALG.APV-210 binding using flow cytometry by gating individual viable cells expressing CD3 and CD8. MFI values (minus FMO) from 2 independent experiments, as well as normalized pooled data from 2 experiments and EC ₅₀ values were determined and plotted using GraphPad Prism 7, non-linear regression of log(agonist) versus normalized response - variable slope. n = 6 donors/group.

results

[00329] In ELISA tests, the binding of biotinylated ALG.APV-210 to CD137 was similar to the binding of non-biotinylated ALG.APV-210 (0.6 nM vs. 0.7 nM). The binding (EC ₅₀ ) of biotinylated ALG.APV-210 to its immunomodulatory target CD137 when expressed on activated primary human and cynomolgus CD8 T cells from CD3-stimulated PBMCs was assessed and compared between the two species as well as with unstimulated cells. As shown using MFI of all CD3+ CD8+ cells in FIG. 22A, 22B and 22C and Table 25, ALG.APV-210 binds to both human and cynomolgus CD8 T cells in a dose-dependent manner, but not to unstimulated cells. Binding to the negative control, ANC107, was low/undetectable for CD3-stimulated or unstimulated human or cynomolgus PBMCs. The maximum amount (MFI) of ALG.APV-210 associated with CD3-stimulated cynomolgus PBMCs was higher compared with human PBMCs, most likely due to a higher proportion of CD3-stimulated cynomolgus PBMCs expressing CD137 or due to an overall higher CD137 expression on cynomolgus CD8 T cells. It is unclear whether cynomolgus cells were more sensitive to stimulation with CD3 compared to human cells, and therefore expressed CD137 to a higher extent, since the cells were stimulated with different clones of CD3 antibody due to the lack of cross-reactivity of the reagents to CD3. CD3 between species.

Table 25. Binding of ALG.APV-210 (EC ₅₀ ) to human and cynomolgus CD8 T cells

ALG.APV-210 binding (nM) Human CD8 T cells (gated from CD3 stimulated PBMCs) Cynomolgus CD8 T cells (gated from CD3-stimulated PBMCs) EC ₅₀ (nM) 0.235 0.236 95% CI (nM) From 0.21 to 0.26 From 0.22 to 0.25

[00330] ALG.APV-210 binding was also assessed by the percentage of CD8 T cells bound by ALG.APV-210. A higher percentage of cynomolgus CD8 T cells bound by ALG.APV-210 was observed compared to human CD8 T cells (71-96% for cynomolgus cells, 65-76% for human cells, see FIG. 23A. 23B and 23C). EC ₅₀ binding values were also calculated from the MFI of CD137-positive cells, resulting in an EC ₅₀ value of 0.17 nM for binding to human CD137 and 0.20 nM for cynomolgus CD137.

[00331] Taken together, the results of the study showed that ALG.APV-210 binds to similar _EC50 values of 0.2 nM to CD3-stimulated human and cynomolgus primary CD8 T cells, but not to unstimulated cells.

Example 15: In vitro activity of anti-5T4-CD137 bispecific antibody in an IFN-γ release assay using human or cynomolgus CD8 T cells in the presence of 5T4

[00332] A study was conducted to compare the functional activity and EC ₅₀ determination of ALG.APV-210 in an agonist activity assay using human or cynomolgus CD8 T cells in the presence of plate-immobilized 5T4.

Materials and methods

[00333] CD8 T cells were suboptimally stimulated with plate-coated anti-CD3 antibodies and activated by cross-linking CD137 via binding of ALG.APV-210 to plate-coated 5T4-Fc. CD8 T cell activation was measured by determining the release of IFN-γ into the cell culture supernatant.

[00334] Isolation and assay design with human CD8 T cells: Human PBMCs were isolated by density gradient centrifugation using Ficoll-Paque (GE Healthcare No. 17-1440-02) from leukocyte concentrates obtained from healthy donors (Clinical Immunology and Transfusion Medicine , Lund, Sweden). CD8+ cells were enriched through negative selection using a CD8+ T cell isolation kit (Miltenyi no. 130-096-495). 96-well assay plates not treated with tissue culture were coated overnight at 4 ºC with 3 μg/ml αCD3, anti-CD3 antibody (clone: OKT-3, Affymetrix eBioscience no. 16-0037-85). The next day, plates were washed in PBS and coated with 5 μg/ml 5T4-Fc for 2 h at 37 °C. Following 5T4-Fc immobilization, assay plates were washed in PBS and blocked for at least 30 min with R10 (RPMI, Gibco #61870010, containing 10% heat-inactivated FBS, Hyclone #SV30160, lot RB35944, and 10 mM Hepes, Gibco # 15630056). Serial dilutions of ALG.APV-210 were prepared in R10 and 50 µl were added in triplicate per assay plate 30 minutes before adding 50 µl of effector cells (enriched CD8 T cells, 1.4 x 10 ⁶ cells/ml or 0.07 × 10 ⁵ cells/well). Assay plates were incubated for 72 h at 37°C and the culture supernatant was collected. IFN-γ levels in the supernatants were measured by ELISA (BD OptiEIA no. 555142). The experiment was performed 4 times, including a total of 8 donors.

[00335] Cynomolgus monkey CD8 T cell isolation and assay design: Cynomolgus monkey (Macaca flavicularis, adult male, 3-15 years) whole blood obtained from Silabe, France was used. Red blood cells were removed by red cell lysis (BD Pharma-lyse, BD Biosciences, #555899) according to the manufacturer's protocol. CD8 ⁺ cells were enriched through positive selection using the CD8 MicroBead kit (Miltenyi Biotec, no. 130-091-112). Plates were coated overnight at 4°C with 1 μg/ml cynomolgus CD3 antibody clone FN-18 (Invitrogen, #APS0301), washed, and coated with 5 μg/ml 5T4-Fc for 2 hours at 37°C. After coating with 5T4-Fc, plates were washed and blocked for at least 30 minutes with R10. ALG.APV-210 was diluted in R10 and 50 µl was added in triplicate to the plates 30 minutes before adding 50 µl CD8 T cells (0.07 x 10 ⁶ cells/well). Assay plates were incubated for 72 h at 37°C and the culture supernatant was collected. IFN-γ levels in the supernatants were measured by ELISA (Monkey IFN-γ ELISA Imaging Kit, MABTECH, no. 3421M-1H-20). The experiment was performed 3 times, including a total of 8 donors. The resulting IFN-γ levels from each human and cynomolgus donor were normalized and mean values were calculated. The mean of normalized IFN-γ levels from all 8 human or cynomolgus donors were pooled and EC ₅₀ was determined by nonlinear regression (log agonist versus normalized response, variable slope) using GraphPad 7.

results

[00336] As shown in FIG. 24 and Table 26, in an assay with human CD8 T cells, the anti-CD137-5T4 bispecific antibody ALG.APV-210 was highly functional with an EC ₅₀ of 0.2 nM. Normalized IFN-γ levels from 4 experiments and a total of 8 donors were pooled and EC50 determined by nonlinear regression using GraphPad 7. The mean and SEM are presented in FIG. 24. Absolute mean IFN-γ values from two donors from one exemplary experiment are presented in FIG. 25A and 25B. The maximum values of CD3 reagents, as well as background levels of IFN-γ, vary from donor to donor within the same experiment. Due to observed differences between donors, data were normalized prior to analysis of the combined data set. Activation of CD8 T cells by ALG.APV-210 was assessed in both the presence and absence of 5T4-Fc to test the dependence on 5T4 cross-linking. The results showed that the anti-CD137-5T4 bispecific antibody ALG.APV-210 induced high T cell activation, as measured by IFN-γ release, when cross-linked to 5T4; in the absence of 5T4 there is no T cell activity (FIGS. 25A and 25B).

[00337] The functional activity of the anti-CD137-5T4 bispecific antibody ALG.APV-210 was also assessed using cynomolgus CD8 T cells. Based on pooled data from 3 experiments and a total of 8 donors, the EC ₅₀ of ALG.APV-210 in the cynomolgus CD8 T cell assay was determined to be 0.4 nM (FIG. 26 and Table 26). Absolute averages from 3 donors from one illustrative experiment are also presented in FIG. 27A, 27B and 27C. Similar to the human cell experiment, absolute values (including maximum values and background levels of IFN-γ) also varied between individual donor cynomolgus monkeys within the same experiment. Therefore, data were normalized prior to EC ₅₀ analysis of the pooled data set. Activation of CD8 T cells by ALG.APV-210 was assessed in both the presence and absence of 5T4-Fc to test the dependence on 5T4-induced CD137 cross-linking. As shown in FIG. 27A-27C, the agonist effect of ALG.APV-210 is 5T4-dependent in the cynomolgus CD8 T cell assay, similar to the result observed in the human cell experiment. In each of FIG. 27A, 27B and 27C show the absolute mean and SEM values for IFN-g for 3 individual donors from one exemplary experiment. Wells without 5T4 were included as a negative control.

Table 26. ALG.APV-210 Binding Assay to Human and Cynomolgus CD8 T Cells

ALG.APV-210 binding (nM) Human CD8 T cells Cynomolgus CD8 T cells EC ₅₀ 0.2 0.4 95%CI 0.17 - 0.21 0.37 - 0.44 Hill slope 2.3 3.4

[00338] The study showed that the EC ₅₀ of the CD137-5T4 bispecific antibody ALG.APV-210 in a functional CD8 T cell activation assay was comparable for humans (0.2 nM) and cynomolgus monkey (0.4 nM). T cell activation was mediated by 5T4-dependent CD137 cross-linking in assays with both human and cynomolgus CD8 T cells.

Example 16. In vitro activity of bispecific antibody to 5T4-CD137 ALG.APV-210 in T cell IFN-γ secretion and T cell proliferation assays

[00339] The agonistic functional activity of the 5T4-CD137 bispecific molecule ALG.APV-210 in inducing target-dependent CD137 activation was assessed in a T cell IFN-γ secretion assay and a T cell proliferation assay using intact peripheral blood mononuclear cells (PBMCs). ). To test the functional activity of CD137 bispecific constructs, primary PBMCs were stimulated with anti-CD3 antibodies to increase the expression of CD137, which is not expressed or is expressed at low levels, on resting T cells. Co-stimulation of primary T cells by TCR and CD137 enhances T cell secretion of the cytokine interferon-gamma (IFN-γ).

Methods

IFN-γ secretion assay:

[00340] Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of healthy individuals using standard Ficoll gradients. The isolated cells were washed in saline buffer to remove platelets. PBMCs were cultured with CHO-K1 cells transfected with human 5T4 or empty vector at approximately 120,000 PBMCs per 30,000 CHO-K1 cells in 96-well plates. Anti-CD3 antibody OKT3 (eBioscience) was added to all wells at a concentration of 1 ng/ml. Cells were cultured in a total volume of 200 μl of RPMI 1640 medium supplemented with 10% fetal bovine serum, sodium pyruvate, antibiotics, and non-essential amino acids. The plates were incubated at 37°C, 5% CO ₂ in humidified incubators for 72 hours. 100 μl of medium was collected after 72 hours of stimulation of PBMC cultures primed with a suboptimal concentration of anti-CD3 antibodies to induce CD137 activation. IFN-γ levels in the supernatant were measured using Milliplex® kits (Millipore) according to the manufacturer's instructions. Data were collected on a Bio-Plex Reader 200 system (Bio-Rad). Nonlinear regression analysis to determine EC ₅₀ values was performed using GraphPad Prism 6® graphing and statistical software.

T cell proliferation assay:

[00341] PBMCs were isolated from the blood of healthy individuals using standard Ficoll gradients. After isolation, cells were washed in saline buffer to remove platelets. PBMCs were labeled with CellTrace ^™ violet (Thermofisher), washed, and cultured with irradiated CHO-K1 cells transfected with human 5T4 at approximately 120,000 PBMCs to 30,000 CHO-K1 cells in 96-well plates. CHO-K1 cells were irradiated (Cell Rad X-ray irradiator, Faxitron Bioptics, LLC) and washed in media before plating with PBMCs. Anti-CD3 antibody OKT3 (eBioscience) was added to all wells at a concentration of 5 ng/ml. Cells were cultured in a total volume of 200 μl of RPMI 1640 medium (GIBCO) supplemented with 10% human serum (SIGMA), sodium pyruvate, antibiotics, and non-essential amino acids. The plates were incubated at 37°C, 5% CO ₂ in humidified incubators for 96 hours.

[00342] Supernatants were removed and cells were labeled in the same assay plates with antibodies to CD4, CD8 and CD5 in PBS buffer with 2% BSA and 2 mM EDTA for 30 minutes on ice. 7AAD (SIGMA) was added to exclude dead cells from the analysis. After washing, the cells were resuspended at a concentration of 120 μl/well, and 70 μl was collected from each well and analyzed in a flow cytometer (LSR-IITM, BD Biosciences). Data analysis was performed using Flowjo software in two ways: by calculating the % of CD8+ live T cell events (CD4- CD8+ CD5+, 7AAD-) that have undergone at least one cell division, or by counting the number of CD8+ live T cell events (CD4- CD8+ CD5+, 7AAD-). In both cases, the analysis was carried out after selecting lymphocytes based on forward (FSC) and side scattering (SSC) light scattering signals. Two parallel trials of each condition were performed and the averages from each duplicate set were plotted using GraphPad Prism 6® graphing and statistical software.

Results and conclusions

[00343] The results of IFN-γ secretion experiments are shown in FIG. 32. Each point on the graphs represents the average of parallel wells. Addition of anti-CD3 antibody alone, without bispecific antibodies, induced very low levels of IFN-γ. Addition of the bispecific molecule ALG.APV-210 increased IFN-γ secretion in a dose-dependent manner. Baseline levels of IFN-γ induced by anti-CD3 antibody, as well as the total amount of secreted IFN-γ induced by ALG.APV-210, varied depending on the PBMC donor (FIG. 32A versus FIG. 32B). Enhanced IFN-γ secretion was observed in the presence of CHO-K1 cells expressing 5T4 but not in the presence of CHO-K1 cells transfected with empty vector control. Therefore, the bispecific molecule ALG.APV-210 requires 5T4 incorporation to stimulate CD137 function as measured by IFN-γ secretion.

[00344] The results of T cell proliferation experiments are shown in FIG. 33. Each point on the graphs represents the average of parallel wells. Polyclonal proliferation of CD8+ T cells was assessed after 96 hours of priming stimulation at a suboptimal concentration of anti-CD3 antibody. Addition of anti-CD3 antibody alone without bispecific antibody induced low levels of CD8+ T cell proliferation. The addition of ALG.APV-210 increased the proliferation of CD8+ T cells in a dose-dependent manner (FIG. 33A and FIG. 33B). Both the percentage of cells undergoing division (upper sections) and the total number of cells (lower sections) collected per well were consistent with sustained cell division and accumulation of dividing CD8+ T cells in the wells. Results were consistent across multiple donors.

Example 17. Functional reporter activity of ALG.APV-210 in the presence of various human tumor cell lines expressing 5T4 protein in a range of densities

[00345] Experiments were conducted to determine whether the ALG.APV-210 construct had binding and functional reporter activity in several human tumor cell lines expressing 5T4 at a range of densities on their surface. The maximum binding capacity of ALG.APV-210 will vary depending on the level of 5T4 tumor antigen expression by cells. Therefore, ALG.APV-210 binding was tested in 3 tumor cell lines (MDA-MB-231, H1975, and TF-1) naturally expressing 5T4 at a range of densities, as well as CHO-K1 cells stably transfected with 5T4 (CHO/5T4) .

Materials and methods

[00346] 5T4 Binding Assay: A 3-fold titer of the ALG.APV-210 construct (ranging from 100 nM to 0.14 nM) was incubated with cell lines expressing human 5T4 for 40 minutes in FACS buffer at 4°C. washed 3 times and detected with PE-labeled secondary goat anti-human Fc antibody (Jackson ImmunoResearch). ALG.APV-210 binding was detected by flow cytometry. All samples were tested in duplicate. Nonlinear regression analysis to determine EC ₅₀ values was performed using GraphPad Prism 6® graphing and statistical software.

[00347] 5T4 Protein Density: The surface density of 5T4 protein present on each cell line was examined using Quantibrite™ beads (BD Pharmingen). Quantibrite™ beads were prepared using the exact cytometer settings used to obtain cells for ALG.APV-210 binding curves. Four populations of beads were gated, and the geometric mean fluorescence value was determined for each peak. A linear regression of Log ₁₀ PE molecules per bead on Log ₁₀ fluorescence was plotted using the equation y = m x + c, where y is equal to Log ₁₀ fluorescence and x is equal to Log ₁₀ PE molecules per bead. From this, the number of PE (5T4) molecules per cell can be calculated. For this particular analysis, the equation was y = 1.036 x - 1.478. All mean fluorescence values were calculated using the geometric mean of the samples as recommended by the manufacturer's protocol.

[00348] CD137 reporter assay: Jurkat/CD137 transfectants carrying a luciferase reporter gene under the control of the NF-κB promoter (Promega) were cultured with H1975, TF-1, MDA-MB-231 or CHO/5T4 tumor cells at a ratio of 30,000 reporter cells per 60,000 target cells in 96-well plates. Fivefold concentrations of the ALG.APV-210 molecule were added, with final concentrations ranging from 10 nM to 0.0006 nM. Cells were cultured in a total volume of 100 μl of RPMI 1640 medium supplemented with 1% fetal bovine serum, sodium pyruvate, antibiotics, and non-essential amino acids. The plates were incubated at 37°C, 5% CO ₂ in humidified incubators for 5 hours. 100 μl of Bio-Glow buffer (Promega) was added to each well, mixed and incubated for 10 minutes before luminescence measurement. Luminescence was measured on a MicroBeta ² 2450 microplate counter (Perkin Elmer). All points represent samples in duplicate. Nonlinear regression analysis to determine EC ₅₀ values was performed using GraphPad Prism 6® graphing and statistical software. Each point on the curve represents the average of parallel wells. The y-axis shows values in relative fluorescence units (RLU).

Results and conclusions

[00349] Each of the tumor cell lines tested differed in the amount of 5T4 protein expressed on the cell surface, as determined using Quantibrite™ beads. In this way, the ability of ALG.APV-210 to bind and be functionally active in the 5T4 expression range can be examined.

[00350] ALG.APV-210 bound to a number of tumor cell lines expressing 5T4 on their surface. As shown in FIG. 34, CHO/5T4 have 10-fold higher 5T4 expression than tumor cell lines endogenously expressing 5T4. Of the three untransfected tumor cell lines, H1975 had the highest 5T4 expression, whereas TF-1 cells had the lowest 5T4 protein density. MDA-MB-231 cells showed intermediate expression of 5T4. Cell surface 5T4 protein densities were calculated from the binding curves, yielding a range of molecules/cell from 17,000 to over 250,000. Table 27 presents cell surface 5T4 protein densities and EC ₅₀ binding values obtained from the binding curves. The expression density of 5T4 on tumor cell lines was calculated using a fluorescence standard.

Table 27. Summary of densities 5T4 and EC ₅₀ ALG.APV-210

Tumor cell line 5T4 molecules/cell EC ₅₀ (nM) CHO/5T4 278000 10 H1975 43000 20 MDA.MB-231 33000 21 TF-1 18000 71

[00351] In FIG. 35 shows the results of a functional reporter assay for CD137. As shown, ALG.APV-210 was sufficiently able to induce NF-κB signaling upon CD137 cross-linking through binding to tumor cell lines CHO/hu5T4, H1975, MDA.MB.231 TF-1, despite the range of 5T4 expression on cell surface from 17,000 to over 250,000. Cell surface 5T4 protein density influenced maximum functional activity (maximum RLU), with CHO/hu5T4 cells exhibiting the highest RLU values. However, three tumor cell lines induced robust reporter functional activity. The results suggest that ALG.APV-210 plays an agonist role in the presence of 5T4-positive cells expressing 5T4 at a range of densities. Table 28 presents the EC ₅₀ values and maximum RLUs induced by ALG.APV-210 in the CD137 reporter assay in the presence of various human 5T4-expressing tumor cell lines.

Table 28. Summary of EC ₅₀ and maximum RLU for ALG.APV-210

Tumor cell line EC ₅₀ (nM) Maximum RLUs CHO/5T4 0.0092 3092200 H1975 0.037 2743132 MDA.MB-231 0.054 2243297 TF-1 0.063 2751522

Example 18: In vitro activity of anti-5T4-CD137 bispecific antibody in an IFN-γ release assay using human CD8 T cells and human HCT116 tumor cells

[00352] The functional activity of the anti-5T4-CD137 bispecific antibody ALG.APV-210 was assessed in a CD8 T cell assay in which CD8+ T cells stimulated with bead-coated anti-CD3 antibodies were cocultured in plates with human HCT116 tumor cells , expressing endogenous levels of 5T4.

Materials and methods

[00353] Mitomycin C treatment of HCT116 cells: HCT116 tumor cells, a human colon carcinoma cell line (ATCC® CCL-247™), expressing endogenous levels of human 5T4 (receptor density = 6.2 x 10 ⁴ /cell, Table 21), pretreated with mitomycin C (0.5 mg/ml, Sigma-Aldrich) at a concentration of 50 μg/ml to inhibit tumor cell overgrowth. After 45 min of incubation with mitomycin C at 37°C, cells were washed three times in R10 medium (RPMI, Gibco no. 61870010, containing 10% heat-inactivated FBS, Hyclone no. SV30160, lot RB35944, and 10 mM Hepes, Gibco no. 15630056), and then re-suspended at a concentration of 10 × 10 ⁶ cells/ml in new R10 medium. 50 μl (5 × 10 ⁵ cells/well) of mitomycin C-treated HCT116 cells were added to tissue culture-treated assay plates (Eppendorf no. 0030 730.119) and incubated at 37°C overnight.

[00354] CD8 T cell assay design: The next day, human PBMCs were isolated by density gradient centrifugation using Ficoll-Paque (GE Healthcare #17-1440-02) from leukocyte concentrates obtained from healthy donors (Clinical Immunology and Transfusion Medicine , Lund, Sweden). CD8+ cells were enriched through negative selection using a CD8+ T cell isolation kit (Miltenyi 130-096-495). Serial dilutions of the ALG.APV-210 bispecific antibody or the Adaptir format isotype control, ANC017 (heavy and light chain variable region sequences for ANC017 are shown below in Tables 29A and 29B) were prepared in R10 medium, and 50 were added to each well of the assay plates. µl and incubated at 37°C for 30 minutes before adding 50 µl of αCD3 beads (4 × 10 ⁸ /ml anti-CD3 antibody clone: OKT-3, Affymetrix eBioscience No. 16-0037-85, coated on beads, diluted to 2 × 10 ⁶ /ml) at a 1:1 ratio of CD8+ T cells to beads, followed by the addition of 50 μl of effector cells (enriched CD8+ cells, 2 × 10 ⁶ /ml or 1 × 10 ⁵ /well). Assay plates were incubated for 72 h at 37°C and the culture supernatant was collected. IFN-γ levels in the supernatants were measured using ELISA (BD OptiEIA no. 555142) and are presented in FIG. 36 in the form of normalized data (mean values and standard deviation). EC ₅₀ values were determined using a non-linear regression of log (agonist) on the normalized response (variable slope) using GraphPad Prism 7. The TOP (maximum functional activity) value was determined by calculating the average of the two highest values of IFN-γ production in the culture supernatant. The experiment was performed 4 times, including a total of 12 donors.

Table 29A. ACN017 variable region sequences

Heavy chain Light chain Amino acid sequence SEQ ID NO: Amino acid sequence SEQ ID NO: CDR1 GFTFSSYA thirty QSISSY 8 CDR2 ISGSGGST 32 A.A.S. 10 CDR3 AKGSSGSYFDL 177 QQYSGYPYT 178 VH/VL EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGSGSYFDLWGQGTLVTVSS 179 DIQMTQSPSFLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSGYPYTFGQGTKLEIK 180

Table 29B. Full length sequence of ACN017

Full length sequence of ACN017 (VH-Fc-VL) SEQ ID NO: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGSGSYFDLWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGS GSGTDFTLTISSLQPEDFATYYCQQYSGYPYTFGQGTKLEIKSSEPKSSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSGGGGSPSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYY CAKGSGSYFDLWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSGYPYTFGQGTKLEIKRS 181

Results and conclusions

[00355] The functional activity of the anti-CD137-5T4 bispecific antibody ALG.APV-210 was determined using human CD8+ T cells (effector cells) cultured in HCT116 cell plates. As shown in FIG. 36, the anti-CD137-5T4 bispecific antibody ALG.APV-210 induced high T cell activation, as measured by IFN-γ release, in a dose-dependent manner in the presence of HCT116 cells expressing endogenous levels of 5T4. For specific EC ₅₀ values, see Table 30, which provides means and 95% CIs for 12 donors from 4 experiments pooled. Endogenous levels of 5T4 expressed by HCT116 cells are sufficient to induce the high agonistic functional activity of ALG.APV-210, as shown in FIG. 37, where IFN-γ levels for 6 individual donors after treatment with ALG.APV-210 are compared with an isotype control (ANC017). Efficacy (maximum IFN-γ response) is also presented in FIG. 38 (fold change from isotype control, mean and SD for 12 donors).

Table 30. EC ₅₀ ALG.APV-210 in CD8+ T cell assay in the presence of HCT116 cells expressing human 5T4

Activity of ALG.APV-210 T cell assay with HCT116 EC ₅₀ (nM) 0.17 95%CI From 0.14 to 0.21

Example 19. In vivo antitumor efficacy of a bispecific antibody in a xenograft model of 5T4-positive tumor HCT-116 in SCID beige mice

[00356] A study was conducted to evaluate the in vivo antitumor efficacy of ALG.APV-210.

Materials and methods

[00357] Female SCID beige mice (6-8 weeks old) from Taconic, Denmark were used in the experiment. All animal procedures were performed in accordance with Swedish legislation on animal rights and protection and with the approval of the Ethical Committee for Animal Experimentation in Lund/Malmö (ethical approval application number: M151-13). PBMCs were isolated from leukocyte concentrates obtained from 4 healthy donors (Clinical Immunology and Transfusion Medicine, Lund, Sweden) by density gradient centrifugation using Ficoll-Paque (GE Healthcare No. 17-1440-02) according to the manufacturer's instructions. The HCT-116 cell line, a human colon carcinoma cell line positive for tumor-associated antigen 5T4, was purchased from ATCC (ATCC® CCL-247™, lot no. 62765668) and cultured according to their recommendations.

[00358] On day 0, log phase HCT-116 tumor cells were inoculated subcutaneously into the right hind flank of mice (3 x 10 ⁶ in 110 μl PBS). The next day (day 1), 7 × 10 ⁶ human PBMCs from 4 donors in 100 μl of PBS were injected intraperitoneally. Intraperitoneal injection of antibodies (100 μl in PBS), starting on day 6, with either vehicle (PBS), 10 μg/mouse ALG.APV-210, or 100 μg/mouse ALG.APV-210, was performed twice per week for a total of 5 injections (n = 5 mice/injection/donor). Tumor growth was monitored and measurements of width, length and height were taken twice a week using calipers, from which tumor volume was calculated (w/2x d/2xh/2x pi x (4/3)). The endpoint of the experiment was tumor volume ≥2 cm ³ , wounded or deteriorated health status of mice. Tumor growth was statistically analyzed using a nonparametric 2-tailed Mann-Whitney T test using GraphPad Prism 7 at different time points (days 9, 13, 16, 20, and 23).

results

[00359] As shown in FIG. 28, ALG.APV-210 at a dose of 10 μg/mouse or 100 μg/mouse demonstrated statistically significant antitumor efficacy (e.g., tumor volume inhibition) compared with the vehicle group from days 13 to 23 (pooled data from 4 donors, For statistical analysis and p-values, see Table 31).

Table 31. Statistical analysis of antitumor efficacy after treatment with ALG.APV-210 in the HCT-116 xenograft model

Days after tumor inoculation Significant difference (p<0.05) in tumor volume between injections, Mann-Whitney, nonparametric 2-sided T-test ALG.APV-210, 10 μg, versus vehicle ALG.APV-210, 100 μg, versus vehicle Day 9 No No Day 13 Yes* p = 0.0109 Yes* p = 0.0262 Day 16 Yes*** p = 0.0008 Yes** p = 0.0010 Day 20 Yes**** p <0.0001 Yes* p = 0.0009 Day 23 Yes** p = 0.0016 Yes* p = 0.0225

[00360] In summary, the study demonstrated that ALG.APV-210 has antitumor properties in a 5T4-positive xenograft model of human colon carcinoma.

Example 20 Pharmacokinetic properties of ALG.APV-209 in BALB/c mice

[00361] A study was conducted to evaluate the pharmacokinetic (PK) properties of ALG.APV-209 and ALG.APV-210 in mice. Normal female BALB/c mice were administered a single dose of 200 μg ALG.APV-209 or ALG.APV-210 intravenously (i.v.) at time 0, and blood was collected from 1-3 mice at each time point. Anesthetized mice were exsanguinated by cardiac puncture and serum was collected at t = 15 min and 2, 6, 24, 48, 72, 96, 168, 336, and 504 h postinjection. Serum concentrations of ALG.APV-209 and ALG.APV-210 were determined using an ELISA method designed to detect intact protein only, using huCD137 ECD-AFH (ALG027) to capture BD to CD137 and biotinylated 5T4 ECD-mFc (ALG029) for BD detection to 5T4. Estimated PK distribution parameters based on non-compartmental analysis (NCA) obtained using Phoenix 64 software (v6.4 WinNonlin™ license) are listed in Table 32, and the estimated half-lives along with fit statistics are shown in FIG. 29 For NCA, a precompiled IV model was used with sparse sampling and equal weighting of metrics.

[00362] In normal female BALB/c mice administered an IV bolus dose of 200 μg of ALG.APV-209 or ALG.APV-210, the apparent terminal half-life determined by NCA was 142 and 215 hours, respectively ( excluding samples that may have been affected by the ADA). The estimated half-life parameters determined using a compartment analysis (2-compartment with IV dosing) were similar for ALG.APV-209 and ALG.APV-210. NCA estimates of clearance and volume for ALG.APV-209 and ALG.APV-210 were 0.263 and 0.204 mL/h/kg and 54 and 63 mL/kg, respectively. ALG.APV-210 had the longest terminal half-life and slightly better clearance values according to both assays. Sudden drops in serum concentrations, indicative of anti-drug antibodies (ADA), were detected at late time points in ALG.APV-210-treated mice, however the presence of ADA could not be confirmed by ELISA methods because the drug was still present in the samples , which also likely resulted in rapid clearance of ADA.

Table 32. PK distribution parameters of ALG.APV-209 and ALG.APV-210 calculated using NCA

FC parameter Units ALG.APV-209 ALG.APV-210 ALG.APV-210 without ADA + serum Rsq 0.82 0.900 0.966 HL lambda Z h 142.0 134.6 215.3 Tmax h 0.25 0.25 0.25 Cmax µg/ml 254.04 252.34 252.34 Cmax SE µg/ml No data 14.6 14.6 Cmax/D kg*mcg/ml/mg 0.025 0.025 0.025 C ₀ µg/ml 262.2 263.6 263.6 AUClast h*mcg/ml 34858 33054 38705 AUCINF_obs h*mcg/ml 38043 36317 49124 AUCINF/D_obs h*kg*mcg/ml/mg 3,804 3.632 4,912 V-obs ml/kg 53.84 53.46 63.22 CL_obs ml/h/kg 0.263 0.275 0.204 VSS/OBS ml/kg 51.1 51.5 62.7 MRTlast h 147.4 136.7 171.9 MRTINF-obs h 194.4 187.2 308.2

- Rsq: Matching statistics for the final hatch phase

- HL lambda z: apparent terminal half-life

- Tmax: time of maximum observed concentration

- Cmax: maximum observed concentration occurring at Tmax

- Cmax SE: root mean square error of data at Tmax (time of maximum average concentration)

- Cmax/D: maximum observed concentration divided by dose

- C ₀ : initial concentration extrapolated to the initial value at time 0

- _obs: based on observed concentrations

- AUClast: area under the curve from dose administration to last measurable concentration

- AUCINF: area under the curve from the moment of dose administration, extrapolated to infinity

- AUCINF/D: AUCINF divided by dose

- V: volume of distribution based on final phase

- CL: serum clearance

- Vss: estimated volume of distribution at steady state

- MRTlast: average residence time to last measurable concentration

- MRTINF: average residence time extrapolated to infinity

Example 21. The Fc part of ALG.APV-210 does not interact with Fcγ receptors

[00363] ALG.APV-210 contains mutations introduced to prevent interaction with Fcγ receptors (FcγR), which can lead to FcγR-mediated T cell activation or ADCC, ADCP, CDC, etc. Therefore, ALG.APV binding -210 was tested in CHO cell transfectants stably expressing the following FcγRs: FcγRI (CD64), FcγRIIa (CD32a His131), FcγRIIb (CD32b), FcγRIIIa (CD16a Val158 and CD16a Phe158), and FcγRIIIb (CD16b). Untransfected CHO cells were used as a negative control. A human IgG1 control molecule was used as a positive control. Various concentrations of ALG.APV-210 or control IgG1 were incubated with FcγR-expressing cell lines, washed, and labeled with a secondary human IgG reagent. The binding of ALG.APV-210 and control IgG1 was determined by flow cytometry.

[00364] The results are presented in FIG. 30A and 30V. In FIG. 30B shows that control IgG1 bound to all FcγR expressing cell lines tested: FcγRI (CD64), FcγRIIa (CD32a His131), FcγRIIb (CD32b), FcγRIIIa (CD16a Val158 and CD16a Phe158) and FcγRIIIb (CD16b), but did not bind to untransfected CHO cells (negative control). Significant binding of control IgG1 to some FcγR-expressing cell lines was observed at concentrations as low as 4 nM. In FIG. 30A shows that there was no detectable binding of the ALG.APV-210 construct to the tested FcγRs: FcγRI (CD64), FcγRIIa (CD32a His131), FcγRIIb (CD32b), FcγRIIIa (CD16a Val158 and CD16a Phe158) and FcγRIIIb (CD16b).

Example 22 In vitro activity of bispecific antibody to 5T4-CD137 in a T cell proliferation assay using human PBMCs and CHO-5T4 cells

[00365] The functional activity of the 5T4-CD137 bispecific molecules ALG.APV-209 and ALG.APV-210 was assessed in a T cell proliferation assay using PBMCs. PBMCs were isolated from the blood of healthy people using standard Ficoll gradients. After isolation, cells were washed in saline buffer to remove platelets. PBMCs were cultured with irradiated human CHO-K1/5T4 cells at approximately 120,000 PBMCs per 30,000 5T4(+) cells in 96-well plates. CHO-K1/5T4 cells were irradiated (Cell Rad X-ray irradiator, Faxitron Bioptics, LLC) and washed in media before plating with PBMCs. Anti-CD3 antibody OKT3 (eBioscience) was added to all wells at a concentration of 1 ng/ml. Cells were cultured in a total volume of 200 μl of RPMI 1640 medium (GIBCO) supplemented with 10% fetal bovine serum (SIGMA), sodium pyruvate, antibiotics, and non-essential amino acids. The plates were incubated at 37 ºC, 5% CO2 in humidified incubators for 72 hours. Supernatants were removed and cells were labeled in the same assay plates with antibodies to CD4, CD8 and CD5 in PBS buffer with 2% BSA and 2 mM EDTA for 30 minutes on ice, 7AAD was added to exclude dead cells. After washing, the cells were resuspended at a concentration of 120 μl/well, and 70 μl was collected from each well in a flow cytometer (LSR-IITM, BD Biosciences). Cell analysis was performed using Flowjo software and analyzed by counting the number of CD4+ or CD8+ live T cell events (CD4+ CD8- CD5+, 7AAD- or CD4- CD8+ CD5+ 7AAD-) after gating of lymphocytes by forward (FSC) and lateral (SSC) signals ) light scattering. Two parallel trials of each condition were performed and the averages from each duplicate set were plotted using GraphPad Prism 6® graphing and statistical software.

[00366] The results of the study are presented in FIG. 31A and 31B. Antigen-specific proliferation of CD8+ T cells was assessed after 72 hours of stimulation of T cells primed with a low concentration of anti-CD3 antibodies. Stimulation with anti-CD3 antibodies induces activation of CD137. Addition of anti-CD3 antibodies alone, without bispecific antibodies, induced some proliferation of CD8+ and CD4+ T cells. Addition of ALG.APV-209 or ALG.APV-210 increased the proliferation of CD8+ T cells (FIG. 31A). In contrast, and as expected based on the selective expression of CD137 on CD8+ T cells, the bispecific antibodies had a limited effect on anti-CD3 antibody-induced CD4+ T cell proliferation (FIG. 31B). The results were consistent among several experiments. A control molecule in Morrison format (ALG.APV-004) was included for comparison.

[00367] Taken together, the results showed that both ALG.APV-209 and ALG.APV-210 enhanced the proliferation of CD8+ T cells. ALG.APV-209 and ALG.APV-210 induced a higher degree of cell proliferation than the Morrison format construct ALG.APV-004.

Example 23 5T4 expression in normal and tumor tissues assessed by immunohistochemical analysis.

[00368] Expression of the 5T4 tumor antigen in normal human tissue and in a number of different tumor types was assessed using immunohistochemistry (IHC).

[00369] Staining Method Development: Five commercially available anti-5T4 antibodies and two in-house developed anti-5T4 antibodies were assessed for 5T4 binding in frozen tissue sections compared to formalin-fixed, paraffin-embedded (FFPE) sections of control cells and test human tissues. Isotype control staining was also included. A mouse monoclonal antibody (clone: MAB4975, R&D Systems) was selected as the antibody with the best performance because it was highly specific and functional in both frozen tissue sections and FFPE sections. The antigen unmasking method and antibody titer were optimized for this antibody.

[00370] Tissues and tissue matrices: Human placental tissue and the mouse colon carcinoma cell line CT26 (from ATCC® CRL-2638) transfected to express human 5T4 were used as positive controls. Human brain, liver, and untransfected CT26 cells were used as negative controls. Human tissues used as positive and negative controls were obtained from the archives of Charles River Laboratories. FFPE tissue microarrays (TMAs) were purchased from US Biomax Inc. All stains were assessed by a research assistant at PAI, Charles River Laboratories.

[00371] IHC staining of normal human tissue microarrays: In a sample of normal human tissues with 24 tissue types from 3 donors (FDA808j-1), 5T4 expression was not detected in any of the major organs.

[00372] Staining for IHC types of human tumor tissue: Screening for 5T4 expression in a matrix of tumor tissues of several organs obtained from 72 patients (FDA808j-2), represented by one core per tumor. 5T4 expression has been detected in tumors of the pancreas, thyroid, breast, liver, uterus, cervix, striated muscle, skin (squamous cell carcinoma), neural tissue (neurofibroma), and bladder.

[00373] 5T4 expression was assessed in TMAs containing tumors from 10-50 patients for each of the selected tumor types shown in Table 33, represented by double or triple nuclei. In bladder cancer, head and neck cancer (H&N), non-small cell lung cancer (NSCLC) and mesothelioma, 50-56% of tumors studied were 5T4 positive. A significant number of pancreatic tumors were also 5T4 positive (36%). 50% of clear cell carcinomas examined were 5T4 positive.

Table 33. 5T4 expression in seven selected tumor types

TMA ID number Total number of donors 5T4+ donors (total number) 5T4+ donors (%) Bladder cancer BL721 18 10 56 Head and neck cancers HN811 19 10 53 Non-small cell lung cancer LC10011a 40 20 50 Mesothelioma MS481c 20 10 50 Pancreas cancer PA721a 22 8 36 Kidney cancer T072b 10 2 20 Ovarian cancer T112c 10 1 10

*****

Incorporation by reference

[00374] All sources, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entirety for all purposes. However, reference to any source, article, publication, patent, patent publication and patent application cited herein is not and should not be construed as an admission or any form of suggestion that it constitutes relevant prior art or constitutes part of the public knowledge in any country in the world.

--->

LIST OF SEQUENCES

<110> Aptevo Research and Development LLC

Alligator Bioscience AB

Bienvenue, David

Misher, Lynda

Hoyos, Gabriela H.

Mitchell, Danielle

Ellmark, Peter

Sall, Anna

Furebring, Christina

Von Schantz, Laura

Fritzell, Sara

Varas, Laura

<120> ONCOFETAL ANTIGEN BINDING PROTEINS AND RELATED

COMPOSITIONS AND METHODS FOR THEM

<130> APVO-057/03WO 327714-2734

<150> US 62/535.017

<151> 2017-07-20

<150> US 62/575,820

<151> 2017-10-23

<150>US 62/648,072

<151> 2018-03-26

<160> 181

<170> PatentIn version 3.5

<210> 1

<211> 24

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragments

<400> 1

ggattcacct tttctcacgg ttct 24

<210> 2

<211> 8

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragments

<400> 2

Gly Phe Thr Phe Ser His Gly Ser

15

<210> 3

<211> 24

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragments

<400> 3

atttcttctg gttctggttc taca 24

<210> 4

<211> 8

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragments

<400> 4

Ile Ser Ser Gly Ser Gly Ser Thr

15

<210> 5

<211> 42

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragments

<400> 5

gcgcgctctt cttactacgg ttcttactac tctattgact at 42

<210> 6

<211> 14

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragments

<400> 6

Ala Arg Ser Ser Tyr Tyr Gly Ser Tyr Tyr Ser Ile Asp Tyr

1 5 10

<210> 7

<211> 18

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragments

<400> 7

cagagcatta gcagctat 18

<210> 8

<211> 6

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragments

<400> 8

Gln Ser Ile Ser Ser Tyr

15

<210> 9

<211> 9

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragments

<400> 9

gctgcatcc 9

<210> 10

<211> 3

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragments

<400> 10

Ala Ala Ser

1

<210> 11

<211> 27

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragments

<400> 11

caacagtact acgacaacct gcccact 27

<210> 12

<211> 9

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragments

<400> 12

Gln Gln Tyr Tyr Asp Asn Leu Pro Thr

15

<210> 13

<211> 363

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragments

<400> 13

gaggtgcagc tgttggagag cgggggaggc ttggtacagc ctggggggtc cctgcgcctc 60

tcctgtgcag ccagcggatt caccttttct cacggttcta tgtactgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcatct atttcttctg gttctggttc tacatactat 180

gcagactccg tgaagggccg gttcaccatc tcccatgaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgcg tgccgaggac acggctgtat attattgtgc gcgctcttct 300

tactacggtt cttactactc tattgactat tggggccagg gaaccctggt caccgtctcc 360

tca 363

<210> 14

<211> 121

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragments

<400> 14

Glu 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 Ser His Gly

20 25 30

Ser Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Gly Ser Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser His 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 Arg Ser Ser Tyr Tyr Gly Ser Tyr Tyr Ser Ile Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 15

<211> 321

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragments

<400> 15

gacatccaga tgacccagtc tccatcctcc ctgagcgcat ctgtaggaga ccgcgtcacc 60

atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

cgtttcagtg gcagtggaag cgggacagat ttcactctca ccatcagcag tctacaacct 240

gaagattttg caacttatta ctgtcaacag tactacgaca acctgcccac ttttggccag 300

gggaccaagc tggagatcaa a 321

<210> 16

<211> 107

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragments

<400> 16

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Asp Asn Leu Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 17

<211> 24

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragments

<400> 17

ggattcacct ttgattacgg ttct 24

<210> 18

<211> 8

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragments

<400> 18

Gly Phe Thr Phe Asp Tyr Gly Ser

15

<210> 19

<211> 363

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragments

<400> 19

gaggtgcagc tgttggagag cgggggaggc ttggtacagc ctggggggtc cctgcgcctc 60

tcctgtgcag ccagcggatt cacctttgat tacggttcta tgtactgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcatct atttcttctg gttctggttc tacatactat 180

gcagactccg tgaagggccg gttcaccatc tcccacgaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgcg tgccgaggac acggctgtat attattgtgc gcgctcttct 300

tactacggtt cttactactc tattgactat tggggccagg gaaccctggt caccgtctcc 360

tca 363

<210> 20

<211> 121

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragments

<400> 20

Glu 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 Gly

20 25 30

Ser Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Gly Ser Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser His 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 Arg Ser Ser Tyr Tyr Gly Ser Tyr Tyr Ser Ile Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 21

<211> 321

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragments

<400> 21

gacatccaga tgacccagtc tccatcctcc ctgagcgcat ctgtaggaga ccgcgtcacc 60

atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcacagtgg ggtcccatca 180

cgtttcagtg gcagtggaag cgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttatta ctgtcaacag tactacgaca acctgcccac ttttggccag 300

gggaccaagc tggagatcaa a 321

<210> 22

<211> 107

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragments

<400> 22

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Asp Asn Leu Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 23

<211> 24

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragments

<400> 23

ggattcacct tttcttacgg ttct 24

<210> 24

<211> 8

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragments

<400> 24

Gly Phe Thr Phe Ser Tyr Gly Ser

15

<210> 25

<211> 363

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragments

<400> 25

gaggtgcagc tgttggagag cgggggaggc ttggtacagc ctggggggtc cctgcgcctc 60

tcctgtgcag ccagcggatt caccttttct tacggttcta tgtactgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcatct atttcttctg gttctggttc tacatactat 180

gcagactccg tgaagggccg gttcaccatc tcccatgaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgcg tgccgaggac acggctgtat attattgtgc gcgctcttct 300

tactacggtt cttactactc tattgactat tggggccagg gaaccctggt caccgtctcc 360

tca 363

<210> 26

<211> 121

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragments

<400> 26

Glu 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 Ser Tyr Gly

20 25 30

Ser Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Gly Ser Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser His 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 Arg Ser Ser Tyr Tyr Gly Ser Tyr Tyr Ser Ile Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 27

<211> 363

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragments

<400> 27

gaggtgcagc tgttggagag cgggggaggc ttggtacagc ctggggggtc cctgcgcctc 60

tcctgtgcag ccagcggatt caccttttct tacggttcta tgtactgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcatct atttcttctg gttctggttc tacatactat 180

gcagactccg tgaagggccg gttcaccatc tcccgtgaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgcg tgccgaggac acggctgtat attattgtgc gcgctcttct 300

tactacggtt cttactactc tattgactat tggggccagg gaaccctggt caccgtctcc 360

tca 363

<210> 28

<211> 121

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragments

<400> 28

Glu 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 Ser Tyr Gly

20 25 30

Ser Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Gly 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 Arg Ser Ser Tyr Tyr Gly Ser Tyr Tyr Ser Ile Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 29

<211> 24

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragment

<400> 29

ggcttcacat tcagcagcta tgct 24

<210> 30

<211> 8

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragment

<400> 30

Gly Phe Thr Phe Ser Ser Tyr Ala

15

<210> 31

<211> 24

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragment

<400> 31

atctccggca gcggcggaag cacc 24

<210> 32

<211> 8

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragment

<400> 32

Ile Ser Gly Ser Gly Gly Ser Thr

15

<210> 33

<211> 42

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragment

<400> 33

gccaggtact atggcggcta ctactccgcc tggatggact ac 42

<210> 34

<211> 14

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragment

<400> 34

Ala Arg Tyr Tyr Gly Gly Tyr Tyr Ser Ala Trp Met Asp Tyr

1 5 10

<210> 35

<211> 27

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragment

<400> 35

cagcagacct atggctacct gcacacc 27

<210> 36

<211> 9

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragment

<400> 36

Gln Gln Thr Tyr Gly Tyr Leu His Thr

15

<210> 37

<211> 363

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragment

<400> 37

gaagtgcagc tgctggagtc cggagggagga ctggtgcagc ctggcggaag cctgaggctg 60

agctgcgctg cctccggctt cacattcagc agctatgcta tgagctgggt gaggcaagcc 120

cctggaaagg gcctggagtg ggtgtccgct atctccggca gcggcggaag cacctactac 180

gctgactccg tcaagggcag gttcaccatc agccgggaca acagcaagaa caccctgtac 240

ctgcagatga atagcctcag ggctgaagac accgctgtgt actactgcgc caggtactat 300

ggcggctact actccgcctg gatggactac tggggacagg gcacactggt gaccgtgtcc 360

agc 363

<210> 38

<211> 121

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragment

<400> 38

Glu 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 Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly 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 Arg Tyr Tyr Gly Gly Tyr Tyr Ser Ala Trp Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 39

<211> 321

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragment

<400> 39

gatattcaga tgacacagtc ccctagctcc ctgtccgcca gcgtgggaga tcgggtgacc 60

atcacctgca gggccagcca gtccatctcc agctatttaa actggtacca gcagaagcct 120

ggaaaggctc ccaagctgct gatctacgcc gcttccagcc tccagagcgg cgtgcctagc 180

aggttctccg gctccggaag cggaacagac ttcaccctga ccatcagctc cctgcagccc 240

gaggactccg ctacctacta ctgccagcag acctatggct acctgcacac cttcggccag 300

ggcacaaagc tggagatcaa g 321

<210> 40

<211> 107

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragment

<400> 40

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ser Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr Gly Tyr Leu His

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 41

<211> 18

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragment

<400> 41

cagtccatct ccagcttc 18

<210> 42

<211> 6

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragment

<400> 42

Gln Ser Ile Ser Ser Phe

15

<210> 43

<211> 321

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragment

<400> 43

gatattcaga tgacacagtc ccctagctcc ctgtccgcca gcgtgggaga tcgggtgacc 60

atcacctgca gggccagcca gtccatctcc agcttcttaa actggtacca gcagaagcct 120

ggaaaggctc ccaagctgct gatctacgcc gcttccagcc tccagagcgg cgtgcctagc 180

aggttctccg gctccggaag cggaacagac ttcaccctga ccatcagctc cctgcagccc 240

gaggactccg ctacctacta ctgccagcag acctatggct acctgcacac cttcggccag 300

ggcacaaagc tggagatcaa g 321

<210> 44

<211> 107

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragment

<400> 44

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Phe

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ser Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr Gly Tyr Leu His

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 45

<211> 363

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragment

<400> 45

gaagtgcagc tgctggagtc cggagggagga ctggtgcagc ctggcggaag cctgaggctg 60

agctgcgctg cctccggctt cacattcagc agctatgcta tgagctgggt gaggcaagcc 120

cctggaaagt gcctggagtg ggtgtccgct atctccggca gcggcggaag cacctactac 180

gctgactccg tcaagggcag gttcaccatc agccgggaca acagcaagaa caccctgtac 240

ctgcagatga atagcctcag ggctgaagac accgctgtgt actactgcgc caggtactat 300

ggcggctact actccgcctg gatggactac tggggacagg gcacactggt gaccgtgtcc 360

agc 363

<210> 46

<211> 121

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragment

<400> 46

Glu 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 Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly 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 Arg Tyr Tyr Gly Gly Tyr Tyr Ser Ala Trp Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 47

<211> 321

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragment

<400> 47

gatattcaga tgacacagtc ccctagctcc ctgtccgcca gcgtgggaga tcgggtgacc 60

atcacctgca gggccagcca gtccatctcc agcttcttaa actggtacca gcagaagcct 120

ggaaaggctc ccaagctgct gatctacgcc gcttccagcc tccagagcgg cgtgcctagc 180

aggttctccg gctccggaag cggaacagac ttcaccctga ccatcagctc cctgcagccc 240

gaggactccg ctacctacta ctgccagcag acctatggct acctgcacac cttcggctgc 300

ggcacaaagc tggagatcaa g 321

<210> 48

<211> 107

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragment

<400> 48

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Phe

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ser Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr Gly Tyr Leu His

85 90 95

Thr Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 49

<211> 321

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragment

<400> 49

gatattcaga tgacacagtc ccctagctcc ctgtccgcca gcgtgggaga tcgggtgacc 60

atcacctgca gggccagcca gtccatctcc agcttcttaa actggtacca gcagaagcct 120

ggaaaggctc ccaagctgct gatctacgcc gcttccagcc tccagagcgg cgtgcctagc 180

aggttctccg gctccggaag cggaacagac ttcaccctga ccatcagctc cctgcagccc 240

gaggacttcg ctacctacta ctgccagcag acctatggct acctgcacac cttcggctgc 300

ggcacaaagc tggagatcaa g 321

<210> 50

<211> 107

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragment

<400> 50

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Phe

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr Gly Tyr Leu His

85 90 95

Thr Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 51

<211> 24

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragment

<400> 51

ggcttcgact tcgagagcta tgct 24

<210> 52

<211> 8

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragment

<400> 52

Gly Phe Asp Phe Glu Ser Tyr Ala

15

<210> 53

<211> 18

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragment

<400> 53

cagtccatca ggagcgcc 18

<210> 54

<211> 6

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragment

<400> 54

Gln Ser Ile Arg Ser Ala

15

<210> 55

<211> 363

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragment

<400> 55

gaagtgcagc tgctggagtc cggagggagga ctggtgcagc ctggcggaag cctgaggctg 60

agctgcgctg cctccggctt cgacttcgag agctatgcta tgagctgggt gaggcaagcc 120

cctggaaagt gcctggagtg ggtgtccgct atctccggca gcggcggaag cacctactac 180

gctgactccg tcaagggcag gttcaccatc agccgggaca acagcaagaa caccctgtac 240

ctgcagatga atagcctcag ggctgaagac accgctgtgt actactgcgc caggtactat 300

ggcggctact actccgcctg gatggactac tggggacagg gcacactggt gaccgtgtcc 360

agc 363

<210> 56

<211> 121

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragment

<400> 56

Glu 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 Asp Phe Glu Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly 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 Arg Tyr Tyr Gly Gly Tyr Tyr Ser Ala Trp Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 57

<211> 321

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragment

<400> 57

gatattcaga tgacacagtc ccctagctcc ctgtccgcca gcgtgggaga tcgggtgacc 60

atcacctgca gggccagcca gtccatcagg agcgccctga actggtacca gcagaagcct 120

ggaaaggctc ccaagctgct gatctacgcc gcttccagcc tccagagcgg cgtgcctagc 180

aggttctccg gctccggaag cggaacagac ttcaccctga ccatcagctc cctgcagccc 240

gaggacttcg ctacctacta ctgccagcag acctatggct acctgcacac cttcggctgc 300

ggcacaaagc tggagatcaa g 321

<210> 58

<211> 107

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragment

<400> 58

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Arg Ser Ala

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr Gly Tyr Leu His

85 90 95

Thr Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 59

<211> 24

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragment

<400> 59

ggcttcgact tcgacagcta tgct 24

<210> 60

<211> 8

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragment

<400> 60

Gly Phe Asp Phe Asp Ser Tyr Ala

15

<210> 61

<211> 24

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragment

<400> 61

atctccggca ggggcggaag cacc 24

<210> 62

<211> 8

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragment

<400> 62

Ile Ser Gly Arg Gly Gly Ser Thr

15

<210> 63

<211> 363

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragment

<400> 63

gaagtgcagc tgctggagtc cggagggagga ctggtgcagc ctggcggaag cctgaggctg 60

agctgcgctg cctccggctt cgacttcgac agctatgcta tgagctgggt gaggcaagcc 120

cctggaaagt gcctggagtg ggtgtccgct atctccggca ggggcggaag cacctactac 180

gctgactccg tcaagggcag gttcaccatc agccgggaca acagcaagaa caccctgtac 240

ctgcagatga atagcctcag ggctgaagac accgctgtgt actactgcgc caggtactat 300

ggcggctact actccgcctg gatggactac tggggacagg gcacactggt gaccgtgtcc 360

agc 363

<210> 64

<211> 121

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragment

<400> 64

Glu 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 Asp Phe Asp Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Arg Gly 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 Arg Tyr Tyr Gly Gly Tyr Tyr Ser Ala Trp Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 65

<211> 321

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragment

<400> 65

gacatccaga tgacccagtc tccatcctcc ctgagcgcat ctgtaggaga ccgcgtcacc 60

atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

cgtttcagtg gcagtggaag cgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttatta ctgtcaacag acttacggtt acctgcacac ttttggctgt 300

gggaccaggc tggagatcaa a 321

<210> 66

<211> 107

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragment

<400> 66

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr Gly Tyr Leu His

85 90 95

Thr Phe Gly Cys Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 67

<211> 321

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragment

<400> 67

gacatccaga tgacccagtc tccatcctcc ctgagcgcat ctgtaggaga ccgcgtcacc 60

atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

cgtttcagtg gcagtggaag cgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttatta ctgtcaacag acttacggtt acctgcacac ttttggccag 300

gggaccaagc tggagatcaa a 321

<210> 68

<211> 107

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragment

<400> 68

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr Gly Tyr Leu His

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 69

<211> 321

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragment

<400> 69

gacatccaga tgacccagtc tccatcctcc ctgagcgcat ctgtaggaga ccgcgtcacc 60

atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

cgtttcagtg gcagtggaag cgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttatta ctgtcaacag acttacggtt acctgcacac ttttggctgc 300

gggaccaagc tggagatcaa a 321

<210> 70

<211> 107

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragment

<400> 70

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr Gly Tyr Leu His

85 90 95

Thr Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 71

<211> 15

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Lab Produced - Hinge/Linker Sequence

<400> 71

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser Ser

1 5 10 15

<210> 72

<211> 15

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Lab Produced - Hinge/Linker Sequence

<400> 72

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Ser Pro Pro Cys Ser

1 5 10 15

<210> 73

<211> 15

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Lab Produced - Hinge/Linker Sequence

<400> 73

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Cys Ser

1 5 10 15

<210> 74

<211> 15

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Lab Produced - Hinge/Linker Sequence

<400> 74

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Ser

1 5 10 15

<210> 75

<211> 15

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Lab Produced - Hinge/Linker Sequence

<400> 75

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Ser Pro Pro Ser Ser

1 5 10 15

<210> 76

<211> 15

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Lab Produced - Hinge/Linker Sequence

<400> 76

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Ser Ser

1 5 10 15

<210> 77

<211> 15

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Lab Produced - Hinge/Linker Sequence

<400> 77

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Ser Pro Pro Cys Ser

1 5 10 15

<210> 78

<211> 15

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Lab Produced - Hinge/Linker Sequence

<400> 78

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Ser Pro Pro Cys Pro

1 5 10 15

<210> 79

<211> 15

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Lab Produced - Hinge/Linker Sequence

<400> 79

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser Pro

1 5 10 15

<210> 80

<211> 15

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Lab Produced - Hinge/Linker Sequence

<400> 80

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Ser Pro Pro Cys Pro

1 5 10 15

<210> 81

<211> 15

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Lab Produced - Hinge/Linker Sequence

<400> 81

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Cys Pro

1 5 10 15

<210> 82

<211> 15

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Lab Produced - Hinge/Linker Sequence

<400> 82

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10 15

<210> 83

<211> 15

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Lab Produced - Hinge/Linker Sequence

<400> 83

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Ser Pro Pro Ser Pro

1 5 10 15

<210> 84

<211> 15

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Lab Produced - Hinge/Linker Sequence

<400> 84

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Ser Pro

1 5 10 15

<210> 85

<211> 6

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Lab Produced - Hinge/Linker Sequence

<400> 85

Ser Cys Pro Pro Cys Pro

15

<210> 86

<211> 20

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Lab Produced - Hinge/Linker Sequence

<400> 86

Asn Tyr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

1 5 10 15

Ser Gly Asn Ser

20

<210> 87

<211> 38

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Lab Produced - Hinge/Linker Sequence

<400> 87

Asn Tyr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

1 5 10 15

Ser Gly Asn Tyr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

20 25 30

Gly Gly Ser Gly Asn Ser

35

<210> 88

<211> 2

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Lab Produced - Hinge/Linker Sequence

<400> 88

Asn Ser

1

<210> 89

<211> 8

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Lab Produced - Hinge/Linker Sequence

<400> 89

Gly Gly Gly Gly Ser Gly Asn Ser

15

<210> 90

<211> 10

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Lab Produced - Hinge/Linker Sequence

<400> 90

Asn Tyr Gly Gly Gly Gly Ser Gly Asn Ser

1 5 10

<210> 91

<211> 13

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Lab Produced - Hinge/Linker Sequence

<400> 91

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Asn Ser

1 5 10

<210> 92

<211> 15

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Lab Produced - Hinge/Linker Sequence

<400> 92

Asn Tyr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Asn Ser

1 5 10 15

<210> 93

<211> 18

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Lab Produced - Hinge/Linker Sequence

<400> 93

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Asn Ser

<210> 94

<211> 8

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Lab Produced - Hinge/Linker Sequence

<400> 94

Gly Cys Pro Pro Cys Pro Asn Ser

15

<210> 95

<211> 5

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Lab Produced - Hinge/Linker Sequence

<400> 95

Gly Gly Gly Gly Ser

15

<210> 96

<211> 15

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Lab Produced - Hinge/Linker Sequence

<400> 96

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 97

<211> 16

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Lab Produced - Hinge/Linker Sequence

<400> 97

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 98

<211> 20

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Lab Produced - Hinge/Linker Sequence

<400> 98

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser

20

<210> 99

<211> 21

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Lab Produced - Hinge/Linker Sequence

<400> 99

Gln Arg His Asn Asn Ser Ser Leu Asn Thr Gly Thr Gln Met Ala Gly

1 5 10 15

His Ser Pro Asn Ser

20

<210> 100

<211> 16

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Lab Produced - Hinge/Linker Sequence

<400> 100

Ser Ser Leu Asn Thr Gly Thr Gln Met Ala Gly His Ser Pro Asn Ser

1 5 10 15

<210> 101

<211> 18

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Lab Produced - Hinge/Linker Sequence

<400> 101

Gln Arg His Asn Asn Ser Ser Leu Asn Thr Gly Thr Gln Met Ala Gly

1 5 10 15

His Ser

<210> 102

<211> 14

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Lab Produced - Hinge/Linker Sequence

<400> 102

Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Ser Pro Asn Ser

1 5 10

<210> 103

<211> 20

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Lab Produced - Hinge/Linker Sequence

<400> 103

Asn Ser Leu Ala Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr

1 5 10 15

Ser Pro Asn Ser

20

<210> 104

<211> 19

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Lab Produced - Hinge/Linker Sequence

<400> 104

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

Pro Asn Ser

<210> 105

<211> 19

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Lab Produced - Hinge/Linker Sequence

<400> 105

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

Pro Gly Ser

<210> 106

<211> 19

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Lab Produced - Hinge/Linker Sequence

<400> 106

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

Pro Ala Ser

<210> 107

<211> 18

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Lab Produced - Hinge/Linker Sequence

<400> 107

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

Pro Ser

<210> 108

<211> 19

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Lab Produced - Hinge/Linker Sequence

<400> 108

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

Pro Ser Ser

<210> 109

<211> 744

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - synthesized scFv sequence

<400> 109

gaggtgcagc tgttggagag cgggggaggc ttggtacagc ctggggggtc cctgcgcctc 60

tcctgtgcag ccagcggatt caccttttct cacggttcta tgtactgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcatct atttcttctg gttctggttc tacatactat 180

gcagactccg tgaagggccg gttcaccatc tcccatgaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgcg tgccgaggac acggctgtat attattgtgc gcgctcttct 300

tactacggtt cttactactc tattgactat tggggccagg gaaccctggt caccgtctcc 360

tcaggtggag gtggctccgg gggtggaggt tccggaggag gcggatcagg tggaggcgga 420

agcgacatcc agatgaccca gtctccatcc tccctgagcg catctgtagg agaccgcgtc 480

accatcactt gccggggcaag tcagagcatt agcagctatt taaattggta tcagcagaaa 540

ccagggaaag cccctaagct cctgatctat gctgcatcca gtttgcaaag tggggtccca 600

tcacgtttca gtggcagtgg aagcgggaca gatttcactc tcaccatcag cagtctacaa 660

cctgaagatt ttgcaactta ttactgtcaa cagtactacg acaacctgcc cacttttggc 720

caggggacca agctggagat caaa 744

<210> 110

<211> 248

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - synthesized scFv sequence

<400> 110

Glu 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 Ser His Gly

20 25 30

Ser Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Gly Ser Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser His 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 Arg Ser Ser Tyr Tyr Gly Ser Tyr Tyr Ser Ile Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln

130 135 140

Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val

145 150 155 160

Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp

165 170 175

Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala

180 185 190

Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser

195 200 205

Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe

210 215 220

Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Asp Asn Leu Pro Thr Phe Gly

225 230 235 240

Gln Gly Thr Lys Leu Glu Ile Lys

245

<210> 111

<211> 744

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - synthesized scFv sequence

<400> 111

gaggtgcagc tgttggagag cgggggaggc ttggtacagc ctggggggtc cctgcgcctc 60

tcctgtgcag ccagcggatt cacctttgat tacggttcta tgtactgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcatct atttcttctg gttctggttc tacatactat 180

gcagactccg tgaagggccg gttcaccatc tcccacgaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgcg tgccgaggac acggctgtat attattgtgc gcgctcttct 300

tactacggtt cttactactc tattgactat tggggccagg gaaccctggt caccgtctcc 360

tcaggtggag gtggctccgg gggtggaggt tccggaggag gcggatcagg tggaggcgga 420

agcgacatcc agatgaccca gtctccatcc tccctgagcg catctgtagg agaccgcgtc 480

accatcactt gccggggcaag tcagagcatt agcagctatt taaattggta tcagcagaaa 540

ccagggaaag cccctaagct cctgatctat gctgcatcca gtttgcacag tggggtccca 600

tcacgtttca gtggcagtgg aagcgggaca gatttcactc tcaccatcag cagtctgcaa 660

cctgaagatt ttgcaactta ttactgtcaa cagtactacg acaacctgcc cacttttggc 720

caggggacca agctggagat caaa 744

<210> 112

<211> 248

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - synthesized scFv sequence

<400> 112

Glu 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 Gly

20 25 30

Ser Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Gly Ser Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser His 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 Arg Ser Ser Tyr Tyr Gly Ser Tyr Tyr Ser Ile Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln

130 135 140

Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val

145 150 155 160

Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp

165 170 175

Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala

180 185 190

Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser

195 200 205

Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe

210 215 220

Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Asp Asn Leu Pro Thr Phe Gly

225 230 235 240

Gln Gly Thr Lys Leu Glu Ile Lys

245

<210> 113

<211> 744

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - synthesized scFv sequence

<400> 113

gaggtgcagc tgttggagag cgggggaggc ttggtacagc ctggggggtc cctgcgcctc 60

tcctgtgcag ccagcggatt caccttttct tacggttcta tgtactgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcatct atttcttctg gttctggttc tacatactat 180

gcagactccg tgaagggccg gttcaccatc tcccatgaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgcg tgccgaggac acggctgtat attattgtgc gcgctcttct 300

tactacggtt cttactactc tattgactat tggggccagg gaaccctggt caccgtctcc 360

tcaggtggag gtggctccgg gggtggaggt tccggaggag gcggatcagg tggaggcgga 420

agcgacatcc agatgaccca gtctccatcc tccctgagcg catctgtagg agaccgcgtc 480

accatcactt gccggggcaag tcagagcatt agcagctatt taaattggta tcagcagaaa 540

ccagggaaag cccctaagct cctgatctat gctgcatcca gtttgcaaag tggggtccca 600

tcacgtttca gtggcagtgg aagcgggaca gatttcactc tcaccatcag cagtctacaa 660

cctgaagatt ttgcaactta ttactgtcaa cagtactacg acaacctgcc cacttttggc 720

caggggacca agctggagat caaa 744

<210> 114

<211> 248

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - synthesized scFv sequence

<400> 114

Glu 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 Ser Tyr Gly

20 25 30

Ser Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Gly Ser Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser His 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 Arg Ser Ser Tyr Tyr Gly Ser Tyr Tyr Ser Ile Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln

130 135 140

Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val

145 150 155 160

Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp

165 170 175

Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala

180 185 190

Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser

195 200 205

Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe

210 215 220

Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Asp Asn Leu Pro Thr Phe Gly

225 230 235 240

Gln Gly Thr Lys Leu Glu Ile Lys

245

<210> 115

<211> 744

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - synthesized scFv sequence

<400> 115

gaggtgcagc tgttggagag cgggggaggc ttggtacagc ctggggggtc cctgcgcctc 60

tcctgtgcag ccagcggatt caccttttct tacggttcta tgtactgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcatct atttcttctg gttctggttc tacatactat 180

gcagactccg tgaagggccg gttcaccatc tcccgtgaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgcg tgccgaggac acggctgtat attattgtgc gcgctcttct 300

tactacggtt cttactactc tattgactat tggggccagg gaaccctggt caccgtctcc 360

tcaggcggcg gcggcagcgg cggcggcggc agcggcggcg gaggctccgg cggcggcggc 420

agcgacatcc agatgaccca gtctccatcc tccctgagcg catctgtagg agaccgcgtc 480

accatcactt gccggggcaag tcagagcatt agcagctatt taaattggta tcagcagaaa 540

ccagggaaag cccctaagct cctgatctat gctgcatcca gtttgcaaag tggggtccca 600

tcacgtttca gtggcagtgg aagcgggaca gatttcactc tcaccatcag cagtctgcaa 660

cctgaagatt ttgcaactta ttactgtcaa cagtactacg acaacctgcc cacttttggc 720

caggggacca agctggagat caaa 744

<210> 116

<211> 248

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - synthesized scFv sequence

<400> 116

Glu 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 Ser Tyr Gly

20 25 30

Ser Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Gly 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 Arg Ser Ser Tyr Tyr Gly Ser Tyr Tyr Ser Ile Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln

130 135 140

Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val

145 150 155 160

Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp

165 170 175

Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala

180 185 190

Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser

195 200 205

Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe

210 215 220

Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Asp Asn Leu Pro Thr Phe Gly

225 230 235 240

Gln Gly Thr Lys Leu Glu Ile Lys

245

<210> 117

<211> 744

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - synthesized scFv sequence

<400> 117

gaagtgcagc tgctggagtc cggagggagga ctggtgcagc ctggcggaag cctgaggctg 60

agctgcgctg cctccggctt cacattcagc agctatgcta tgagctgggt gaggcaagcc 120

cctggaaagg gcctggagtg ggtgtccgct atctccggca gcggcggaag cacctactac 180

gctgactccg tcaagggcag gttcaccatc agccgggaca acagcaagaa caccctgtac 240

ctgcagatga atagcctcag ggctgaagac accgctgtgt actactgcgc caggtactat 300

ggcggctact actccgcctg gatggactac tggggacagg gcacactggt gaccgtgtcc 360

agcggcggag gcggctccgg aggcggtggc tccggaggag gcggaagcgg aggaggaggc 420

tccgatattc agatgacaca gtcccctagc tccctgtccg ccagcgtggg agatcgggtg 480

accatcacct gcagggccag ccagtccatc tccagctatt taaactggta ccagcagaag 540

cctggaaagg ctcccaagct gctgatctac gccgcttcca gcctccagag cggcgtgcct 600

agcaggttct ccggctccgg aagcggaaca gacttcaccc tgaccatcag ctccctgcag 660

cccgaggact ccgctaccta ctactgccag cagacctatg gctacctgca caccttcggc 720

cagggcacaa agctggagat caag 744

<210> 118

<211> 248

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - synthesized scFv sequence

<400> 118

Glu 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 Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly 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 Arg Tyr Tyr Gly Gly Tyr Tyr Ser Ala Trp Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln

130 135 140

Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val

145 150 155 160

Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp

165 170 175

Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala

180 185 190

Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser

195 200 205

Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Ser

210 215 220

Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr Gly Tyr Leu His Thr Phe Gly

225 230 235 240

Gln Gly Thr Lys Leu Glu Ile Lys

245

<210> 119

<211> 744

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - synthesized scFv sequence

<400> 119

gaagtgcagc tgctggagtc cggagggagga ctggtgcagc ctggcggaag cctgaggctg 60

agctgcgctg cctccggctt cacattcagc agctatgcta tgagctgggt gaggcaagcc 120

cctggaaagg gcctggagtg ggtgtccgct atctccggca gcggcggaag cacctactac 180

gctgactccg tcaagggcag gttcaccatc agccgggaca acagcaagaa caccctgtac 240

ctgcagatga atagcctcag ggctgaagac accgctgtgt actactgcgc caggtactat 300

ggcggctact actccgcctg gatggactac tggggacagg gcacactggt gaccgtgtcc 360

agcggcggag gcggctccgg aggcggtggc tccggaggag gcggaagcgg aggaggaggc 420

tccgatattc agatgacaca gtcccctagc tccctgtccg ccagcgtggg agatcgggtg 480

accatcacct gcagggccag ccagtccatc tccagcttct taaactggta ccagcagaag 540

cctggaaagg ctcccaagct gctgatctac gccgcttcca gcctccagag cggcgtgcct 600

agcaggttct ccggctccgg aagcggaaca gacttcaccc tgaccatcag ctccctgcag 660

cccgaggact ccgctaccta ctactgccag cagacctatg gctacctgca caccttcggc 720

cagggcacaa agctggagat caag 744

<210> 120

<211> 248

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - synthesized scFv sequence

<400> 120

Glu 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 Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly 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 Arg Tyr Tyr Gly Gly Tyr Tyr Ser Ala Trp Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln

130 135 140

Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val

145 150 155 160

Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Phe Leu Asn Trp

165 170 175

Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala

180 185 190

Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser

195 200 205

Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Ser

210 215 220

Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr Gly Tyr Leu His Thr Phe Gly

225 230 235 240

Gln Gly Thr Lys Leu Glu Ile Lys

245

<210> 121

<211> 744

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - synthesized scFv sequence

<400> 121

gaagtgcagc tgctggagtc cggagggagga ctggtgcagc ctggcggaag cctgaggctg 60

agctgcgctg cctccggctt cacattcagc agctatgcta tgagctgggt gaggcaagcc 120

cctggaaagt gcctggagtg ggtgtccgct atctccggca gcggcggaag cacctactac 180

gctgactccg tcaagggcag gttcaccatc agccgggaca acagcaagaa caccctgtac 240

ctgcagatga atagcctcag ggctgaagac accgctgtgt actactgcgc caggtactat 300

ggcggctact actccgcctg gatggactac tggggacagg gcacactggt gaccgtgtcc 360

agcggcggag gcggctccgg aggcggtggc tccggaggag gcggaagcgg aggaggaggc 420

tccgatattc agatgacaca gtcccctagc tccctgtccg ccagcgtggg agatcgggtg 480

accatcacct gcagggccag ccagtccatc tccagcttct taaactggta ccagcagaag 540

cctggaaagg ctcccaagct gctgatctac gccgcttcca gcctccagag cggcgtgcct 600

agcaggttct ccggctccgg aagcggaaca gacttcaccc tgaccatcag ctccctgcag 660

cccgaggact ccgctaccta ctactgccag cagacctatg gctacctgca caccttcggc 720

tgcggcacaa agctggagat caag 744

<210> 122

<211> 248

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - synthesized scFv sequence

<400> 122

Glu 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 Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly 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 Arg Tyr Tyr Gly Gly Tyr Tyr Ser Ala Trp Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln

130 135 140

Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val

145 150 155 160

Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Phe Leu Asn Trp

165 170 175

Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala

180 185 190

Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser

195 200 205

Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Ser

210 215 220

Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr Gly Tyr Leu His Thr Phe Gly

225 230 235 240

Cys Gly Thr Lys Leu Glu Ile Lys

245

<210> 123

<211> 744

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - synthesized scFv sequence

<400> 123

gaagtgcagc tgctggagtc cggagggagga ctggtgcagc ctggcggaag cctgaggctg 60

agctgcgctg cctccggctt cacattcagc agctatgcta tgagctgggt gaggcaagcc 120

cctggaaagt gcctggagtg ggtgtccgct atctccggca gcggcggaag cacctactac 180

gctgactccg tcaagggcag gttcaccatc agccgggaca acagcaagaa caccctgtac 240

ctgcagatga atagcctcag ggctgaagac accgctgtgt actactgcgc caggtactat 300

ggcggctact actccgcctg gatggactac tggggacagg gcacactggt gaccgtgtcc 360

agcggcggag gcggctccgg aggcggtggc tccggaggag gcggaagcgg aggaggaggc 420

tccgatattc agatgacaca gtcccctagc tccctgtccg ccagcgtggg agatcgggtg 480

accatcacct gcagggccag ccagtccatc tccagcttct taaactggta ccagcagaag 540

cctggaaagg ctcccaagct gctgatctac gccgcttcca gcctccagag cggcgtgcct 600

agcaggttct ccggctccgg aagcggaaca gacttcaccc tgaccatcag ctccctgcag 660

cccgaggact tcgctaccta ctactgccag cagacctatg gctacctgca caccttcggc 720

tgcggcacaa agctggagat caag 744

<210> 124

<211> 248

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - synthesized scFv sequence

<400> 124

Glu 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 Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly 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 Arg Tyr Tyr Gly Gly Tyr Tyr Ser Ala Trp Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln

130 135 140

Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val

145 150 155 160

Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Phe Leu Asn Trp

165 170 175

Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala

180 185 190

Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser

195 200 205

Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe

210 215 220

Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr Gly Tyr Leu His Thr Phe Gly

225 230 235 240

Cys Gly Thr Lys Leu Glu Ile Lys

245

<210> 125

<211> 744

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - synthesized scFv sequence

<400> 125

gaagtgcagc tgctggagtc cggagggagga ctggtgcagc ctggcggaag cctgaggctg 60

agctgcgctg cctccggctt cgacttcgag agctatgcta tgagctgggt gaggcaagcc 120

cctggaaagt gcctggagtg ggtgtccgct atctccggca gcggcggaag cacctactac 180

gctgactccg tcaagggcag gttcaccatc agccgggaca acagcaagaa caccctgtac 240

ctgcagatga atagcctcag ggctgaagac accgctgtgt actactgcgc caggtactat 300

ggcggctact actccgcctg gatggactac tggggacagg gcacactggt gaccgtgtcc 360

agcggcggag gcggctccgg aggcggtggc tccggaggag gcggaagcgg aggaggaggc 420

tccgatattc agatgacaca gtcccctagc tccctgtccg ccagcgtggg agatcgggtg 480

accatcacct gcagggccag ccagtccatc aggagcgccc tgaactggta ccagcagaag 540

cctggaaagg ctcccaagct gctgatctac gccgcttcca gcctccagag cggcgtgcct 600

agcaggttct ccggctccgg aagcggaaca gacttcaccc tgaccatcag ctccctgcag 660

cccgaggact tcgctaccta ctactgccag cagacctatg gctacctgca caccttcggc 720

tgcggcacaa agctggagat caag 744

<210> 126

<211> 248

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - synthesized scFv sequence

<400> 126

Glu 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 Asp Phe Glu Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly 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 Arg Tyr Tyr Gly Gly Tyr Tyr Ser Ala Trp Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln

130 135 140

Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val

145 150 155 160

Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Arg Ser Ala Leu Asn Trp

165 170 175

Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala

180 185 190

Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser

195 200 205

Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe

210 215 220

Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr Gly Tyr Leu His Thr Phe Gly

225 230 235 240

Cys Gly Thr Lys Leu Glu Ile Lys

245

<210> 127

<211> 744

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - synthesized scFv sequence

<400> 127

gaagtgcagc tgctggagtc cggagggagga ctggtgcagc ctggcggaag cctgaggctg 60

agctgcgctg cctccggctt cgacttcgac agctatgcta tgagctgggt gaggcaagcc 120

cctggaaagt gcctggagtg ggtgtccgct atctccggca ggggcggaag cacctactac 180

gctgactccg tcaagggcag gttcaccatc agccgggaca acagcaagaa caccctgtac 240

ctgcagatga atagcctcag ggctgaagac accgctgtgt actactgcgc caggtactat 300

ggcggctact actccgcctg gatggactac tggggacagg gcacactggt gaccgtgtcc 360

agcggcggag gcggctccgg aggcggtggc tccggaggag gcggaagcgg aggaggaggc 420

tccgatattc agatgacaca gtcccctagc tccctgtccg ccagcgtggg agatcgggtg 480

accatcacct gcagggccag ccagtccatc aggagcgccc tgaactggta ccagcagaag 540

cctggaaagg ctcccaagct gctgatctac gccgcttcca gcctccagag cggcgtgcct 600

agcaggttct ccggctccgg aagcggaaca gacttcaccc tgaccatcag ctccctgcag 660

cccgaggact tcgctaccta ctactgccag cagacctatg gctacctgca caccttcggc 720

tgcggcacaa agctggagat caag 744

<210> 128

<211> 248

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - synthesized scFv sequence

<400> 128

Glu 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 Asp Phe Asp Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Arg Gly 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 Arg Tyr Tyr Gly Gly Tyr Tyr Ser Ala Trp Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln

130 135 140

Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val

145 150 155 160

Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Arg Ser Ala Leu Asn Trp

165 170 175

Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala

180 185 190

Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser

195 200 205

Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe

210 215 220

Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr Gly Tyr Leu His Thr Phe Gly

225 230 235 240

Cys Gly Thr Lys Leu Glu Ile Lys

245

<210> 129

<211> 744

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - synthesized scFv sequence

<400> 129

gaggtgcagc tgttggagag cgggggaggc ttggtacagc ctggggggtc cctgcgcctc 60

tcctgtgcag ccagcggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactat 180

gcagactccg tgaagggccg gttcaccatc tcccgtgaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgcg tgccgaggac acggctgtat attattgtgc gcgctactac 300

ggtggttact actctgcttg gatggactat tggggccagg gaaccctggt caccgtctcc 360

tcaggcggtg gaggcagcgg tggggggtggg tctggaggcg gtggcagtgg cggcggaggc 420

tctgacatcc agatgaccca gtctccatcc tccctgagcg catctgtagg agaccgcgtc 480

accatcactt gccggggcaag tcagagcatt agcagctatt taaattggta tcagcagaaa 540

ccagggaaag cccctaagct cctgatctat gctgcatcca gtttgcaaag tggggtccca 600

tcacgtttca gtggcagtgg aagcgggaca gatttcactc tcaccatcag cagtctgcaa 660

cctgaagatt ttgcaactta ttactgtcaa cagacttacg gttacctgca cacttttggc 720

caggggacca agctggagat caaa 744

<210> 130

<211> 248

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - synthesized scFv sequence

<400> 130

Glu 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 Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly 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 Arg Tyr Tyr Gly Gly Tyr Tyr Ser Ala Trp Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln

130 135 140

Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val

145 150 155 160

Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp

165 170 175

Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala

180 185 190

Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser

195 200 205

Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe

210 215 220

Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr Gly Tyr Leu His Thr Phe Gly

225 230 235 240

Gln Gly Thr Lys Leu Glu Ile Lys

245

<210> 131

<211> 744

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - synthesized scFv sequence

<400> 131

gaggtgcagc tgttggagag cgggggaggc ttggtacagc ctggggggtc cctgcgcctc 60

tcctgtgcag ccagcggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120

ccagggaagt gcctggagtg ggtctcagct attagtggta gtggtggtag cacatactat 180

gcagactccg tgaagggccg gttcaccatc tcccgtgaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgcg tgccgaggac acggctgtat attattgtgc gcgctactac 300

ggtggttact actctgcttg gatggactat tggggccagg gaaccctggt caccgtctcc 360

tcaggcggtg gaggcagcgg tggggggtggg tctggaggcg gtggcagtgg cggcggaggc 420

tctgacatcc agatgaccca gtctccatcc tccctgagcg catctgtagg agaccgcgtc 480

accatcactt gccggggcaag tcagagcatt agcagctatt taaattggta tcagcagaaa 540

ccagggaaag cccctaagct cctgatctat gctgcatcca gtttgcaaag tggggtccca 600

tcacgtttca gtggcagtgg aagcgggaca gatttcactc tcaccatcag cagtctgcaa 660

cctgaagatt ttgcaactta ttactgtcaa cagacttacg gttacctgca cacttttggc 720

tgcgggacca agctggagat caaa 744

<210> 132

<211> 248

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - synthesized scFv sequence

<400> 132

Glu 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 Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly 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 Arg Tyr Tyr Gly Gly Tyr Tyr Ser Ala Trp Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln

130 135 140

Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val

145 150 155 160

Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp

165 170 175

Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala

180 185 190

Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser

195 200 205

Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe

210 215 220

Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr Gly Tyr Leu His Thr Phe Gly

225 230 235 240

Cys Gly Thr Lys Leu Glu Ile Lys

245

<210> 133

<211> 744

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - synthesized scFv sequence

<400> 133

gacatccaga tgacccagtc tccatcctcc ctgagcgcat ctgtaggaga ccgcgtcacc 60

atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

cgtttcagtg gcagtggaag cgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttatta ctgtcaacag acttacggtt acctgcacac ttttggctgc 300

gggaccaagc tggagatcaa aggcggtgga ggcagcggtg ggggtgggtc tggaggcggt 360

ggcagtggcg gcggaggctc tgaggtgcag ctgttggaga gcgggggagg cttggtacag 420

cctggggggt ccctgcgcct ctcctgtgca gccagcggat tcacctttag cagctatgcc 480

atgagctggg tccgccaggc tccagggaag tgcctggagt gggtctcagc tattagtggt 540

agtggtggta gcacatacta tgcagactcc gtgaagggcc ggttcaccat ctcccgtgac 600

aattccaaga acacgctgta tctgcaaatg aacagcctgc gtgccgagga cacggctgta 660

tattattgtg cgcgctacta cggtggttac tactctgctt ggatggacta ttggggccag 720

ggaaccctgg tcaccgtctc ctca 744

<210> 134

<211> 248

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - synthesized scFv sequence

<400> 134

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr Gly Tyr Leu His

85 90 95

Thr Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

115 120 125

Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser

130 135 140

Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala

145 150 155 160

Met Ser Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Val Ser

165 170 175

Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys

180 185 190

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu

195 200 205

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

210 215 220

Arg Tyr Tyr Gly Gly Tyr Tyr Ser Ala Trp Met Asp Tyr Trp Gly Gln

225 230 235 240

Gly Thr Leu Val Thr Val Ser Ser

245

<210> 135

<211> 2304

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - polynucleotide construct for 5T4 x 4-1BB

<400> 135

atggaagcac cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccggt 60

gaggtgcagc tgttggagag cgggggaggc ttggtacagc ctggggggtc cctgcgcctc 120

tcctgtgcag ccagcggatt caccttttct cacggttcta tgtactgggt ccgccaggct 180

ccagggaagg ggctggagtg ggtctcatct atttcttctg gttctggttc tacatactat 240

gcagactccg tgaagggccg gttcaccatc tcccatgaca attccaagaa cacgctgtat 300

ctgcaaatga acagcctgcg tgccgaggac acggctgtat attattgtgc gcgctcttct 360

tactacggtt cttactactc tattgactat tggggccagg gaaccctggt caccgtctcc 420

tcaggtggag gtggctccgg gggtggaggt tccggaggag gcggatcagg tggaggcgga 480

agcgacatcc agatgaccca gtctccatcc tccctgagcg catctgtagg agaccgcgtc 540

accatcactt gccgggcaag tcagagcatt agcagctatt taaattggta tcagcagaaa 600

ccagggaaag cccctaagct cctgatctat gctgcatcca gtttgcaaag tggggtccca 660

tcacgtttca gtggcagtgg aagcgggaca gatttcactc tcaccatcag cagtctacaa 720

cctgaagatt ttgcaactta ttactgtcaa cagtactacg acaacctgcc cacttttggc 780

caggggacca agctggagat caaatcgagt gagcccaaat cttctgacaa aactcacaca 840

tgcccaccgt gcccagcacc tgaagccgcg ggtgcaccgt cagtcttcct cttcccccca 900

aaacccaagg acaccctcat gatctcccgg acccctgagg tcacatgcgt ggtggtggac 960

gtgagccacg aagaccctga ggtcaagttc aactggtacg tggacggcgt ggaggtgcat 1020

aatgccaaga caaagccgcg ggagggagcag tacaacagca cgtaccgtgt ggtcagcgtc 1080

ctcaccgtcc tgcaccagga ctggctgaat ggcaaggaat acaagtgcgc ggtctccaac 1140

aaagccctcc cagcccccat cgagaaaacc atctccaaag ccaaagggca gccccgagaa 1200

ccacaggtgt acaccctgcc cccacccgg gatgagctga ccaagaacca ggtcagcctg 1260

acctgcctgg tcaaaggctt ctatccaagc gacatcgccg tggagtggga gagcaatggg 1320

cagccggaga acaactacaa gaccacgcct cccgtgctgg actccgacgg ctccttcttc 1380

ctctacagca agctcaccgt ggacaagagc aggtggcagc aggggaacgt cttctcatgc 1440

tccgtgatgc atgaggctct gcacaaccac tacacgcaga agagcctctc cctgtctccg 1500

ggttccggag gtggcggttc gggaggtggc gggtcaggag gtgggggatc cccttcagaa 1560

gtgcagctgc tggagtccgg aggaggactg gtgcagcctg gcggaagcct gaggctgagc 1620

tgcgctgcct ccggcttcac attcagcagc tatgctatga gctgggtgag gcaagcccct 1680

ggaaagggcc tggagtgggt gtccgctatc tccggcagcg gcggaagcac ctactacgct 1740

gactccgtca agggcaggtt caccatcagc cgggacaaca gcaagaacac cctgtacctg 1800

cagatgaata gcctcagggc tgaagacacc gctgtgtact actgcgccag gtactatggc 1860

ggctactact ccgcctggat ggactactgg ggacagggca cactggtgac cgtgtccagc 1920

ggcggaggcg gctccggagg cggtggctcc ggaggaggcg gaagcggagg aggaggctcc 1980

gatattcaga tgacacagtc ccctagctcc ctgtccgcca gcgtgggaga tcgggtgacc 2040

atcacctgca gggccagcca gtccatctcc agctatttaa actggtacca gcagaagcct 2100

ggaaaggctc ccaagctgct gatctacgcc gcttccagcc tccagagcgg cgtgcctagc 2160

aggttctccg gctccggaag cggaacagac ttcaccctga ccatcagctc cctgcagccc 2220

gaggactccg ctacctacta ctgccagcag acctatggct acctgcacac cttcggccag 2280

ggcacaaagc tggagatcaa gcgc 2304

<210> 136

<211> 768

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - polypeptide construct to 5T4 x 4-1BB

<400> 136

Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro

1 5 10 15

Asp Thr Thr Gly Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val

20 25 30

Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr

35 40 45

Phe Ser His Gly Ser Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly

50 55 60

Leu Glu Trp Val Ser Ser Ile Ser Ser Gly Ser Gly Ser Thr Tyr Tyr

65 70 75 80

Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser His Asp Asn Ser Lys

85 90 95

Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala

100 105 110

Val Tyr Tyr Cys Ala Arg Ser Ser Tyr Tyr Gly Ser Tyr Tyr Ser Ile

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

145 150 155 160

Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val

165 170 175

Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser

180 185 190

Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu

195 200 205

Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser

210 215 220

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln

225 230 235 240

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Asp Asn Leu

245 250 255

Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Ser Ser Glu Pro

260 265 270

Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

275 280 285

Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

290 295 300

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

305 310 315 320

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

325 330 335

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

340 345 350

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

355 360 365

Leu Asn Gly Lys Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala Leu Pro

370 375 380

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

385 390 395 400

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn

405 410 415

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

420 425 430

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

435 440 445

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

450 455 460

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

465 470 475 480

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

485 490 495

Ser Leu Ser Pro Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

500 505 510

Gly Gly Gly Gly Ser Pro Ser Glu Val Gln Leu Leu Glu Ser Gly Gly

515 520 525

Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser

530 535 540

Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro

545 550 555 560

Gly Lys Gly Leu Glu Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser

565 570 575

Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

580 585 590

Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu

595 600 605

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Gly Gly Tyr Tyr Ser

610 615 620

Ala Trp Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

625 630 635 640

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

645 650 655

Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser

660 665 670

Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser

675 680 685

Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

690 695 700

Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser

705 710 715 720

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

725 730 735

Ser Leu Gln Pro Glu Asp Ser Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr

740 745 750

Gly Tyr Leu His Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg

755 760 765

<210> 137

<211> 2304

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - polynucleotide construct for 5T4 x 4-1BB

<400> 137

atggaagcac cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccggt 60

gaggtgcagc tgttggagag cgggggaggc ttggtacagc ctggggggtc cctgcgcctc 120

tcctgtgcag ccagcggatt caccttttct cacggttcta tgtactgggt ccgccaggct 180

ccagggaagg ggctggagtg ggtctcatct atttcttctg gttctggttc tacatactat 240

gcagactccg tgaagggccg gttcaccatc tcccatgaca attccaagaa cacgctgtat 300

ctgcaaatga acagcctgcg tgccgaggac acggctgtat attattgtgc gcgctcttct 360

tactacggtt cttactactc tattgactat tggggccagg gaaccctggt caccgtctcc 420

tcaggtggag gtggctccgg gggtggaggt tccggaggag gcggatcagg tggaggcgga 480

agcgacatcc agatgaccca gtctccatcc tccctgagcg catctgtagg agaccgcgtc 540

accatcactt gccgggcaag tcagagcatt agcagctatt taaattggta tcagcagaaa 600

ccagggaaag cccctaagct cctgatctat gctgcatcca gtttgcaaag tggggtccca 660

tcacgtttca gtggcagtgg aagcgggaca gatttcactc tcaccatcag cagtctacaa 720

cctgaagatt ttgcaactta ttactgtcaa cagtactacg acaacctgcc cacttttggc 780

caggggacca agctggagat caaatcgagt gagcccaaat cttctgacaa aactcacaca 840

tgcccaccgt gcccagcacc tgaagccgcg ggtgcaccgt cagtcttcct cttcccccca 900

aaacccaagg acaccctcat gatctcccgg acccctgagg tcacatgcgt ggtggtggac 960

gtgagccacg aagaccctga ggtcaagttc aactggtacg tggacggcgt ggaggtgcat 1020

aatgccaaga caaagccgcg ggagggagcag tacaacagca cgtaccgtgt ggtcagcgtc 1080

ctcaccgtcc tgcaccagga ctggctgaat ggcaaggaat acaagtgcgc ggtctccaac 1140

aaagccctcc cagcccccat cgagaaaacc atctccaaag ccaaagggca gccccgagaa 1200

ccacaggtgt acaccctgcc cccacccgg gatgagctga ccaagaacca ggtcagcctg 1260

acctgcctgg tcaaaggctt ctatccaagc gacatcgccg tggagtggga gagcaatggg 1320

cagccggaga acaactacaa gaccacgcct cccgtgctgg actccgacgg ctccttcttc 1380

ctctacagca agctcaccgt ggacaagagc aggtggcagc aggggaacgt cttctcatgc 1440

tccgtgatgc atgaggctct gcacaaccac tacacgcaga agagcctctc cctgtctccg 1500

ggttccggag gtggcggttc gggaggtggc gggtcaggag gtgggggatc cccttcagaa 1560

gtgcagctgc tggagtccgg aggaggactg gtgcagcctg gcggaagcct gaggctgagc 1620

tgcgctgcct ccggcttcac attcagcagc tatgctatga gctgggtgag gcaagcccct 1680

ggaaagggcc tggagtgggt gtccgctatc tccggcagcg gcggaagcac ctactacgct 1740

gactccgtca agggcaggtt caccatcagc cgggacaaca gcaagaacac cctgtacctg 1800

cagatgaata gcctcagggc tgaagacacc gctgtgtact actgcgccag gtactatggc 1860

ggctactact ccgcctggat ggactactgg ggacagggca cactggtgac cgtgtccagc 1920

ggcggaggcg gctccggagg cggtggctcc ggaggaggcg gaagcggagg aggaggctcc 1980

gatattcaga tgacacagtc ccctagctcc ctgtccgcca gcgtgggaga tcgggtgacc 2040

atcacctgca gggccagcca gtccatctcc agcttcttaa actggtacca gcagaagcct 2100

ggaaaggctc ccaagctgct gatctacgcc gcttccagcc tccagagcgg cgtgcctagc 2160

aggttctccg gctccggaag cggaacagac ttcaccctga ccatcagctc cctgcagccc 2220

gaggactccg ctacctacta ctgccagcag acctatggct acctgcacac cttcggccag 2280

ggcacaaagc tggagatcaa gcgc 2304

<210> 138

<211> 768

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - polypeptide construct to 5T4 x 4-1BB

<400> 138

Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro

1 5 10 15

Asp Thr Thr Gly Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val

20 25 30

Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr

35 40 45

Phe Ser His Gly Ser Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly

50 55 60

Leu Glu Trp Val Ser Ser Ile Ser Ser Gly Ser Gly Ser Thr Tyr Tyr

65 70 75 80

Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser His Asp Asn Ser Lys

85 90 95

Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala

100 105 110

Val Tyr Tyr Cys Ala Arg Ser Ser Tyr Tyr Gly Ser Tyr Tyr Ser Ile

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

145 150 155 160

Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val

165 170 175

Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser

180 185 190

Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu

195 200 205

Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser

210 215 220

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln

225 230 235 240

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Asp Asn Leu

245 250 255

Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Ser Ser Glu Pro

260 265 270

Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

275 280 285

Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

290 295 300

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

305 310 315 320

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

325 330 335

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

340 345 350

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

355 360 365

Leu Asn Gly Lys Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala Leu Pro

370 375 380

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

385 390 395 400

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn

405 410 415

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

420 425 430

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

435 440 445

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

450 455 460

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

465 470 475 480

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

485 490 495

Ser Leu Ser Pro Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

500 505 510

Gly Gly Gly Gly Ser Pro Ser Glu Val Gln Leu Leu Glu Ser Gly Gly

515 520 525

Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser

530 535 540

Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro

545 550 555 560

Gly Lys Gly Leu Glu Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser

565 570 575

Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

580 585 590

Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu

595 600 605

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Gly Gly Tyr Tyr Ser

610 615 620

Ala Trp Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

625 630 635 640

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

645 650 655

Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser

660 665 670

Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser

675 680 685

Ile Ser Ser Phe Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

690 695 700

Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser

705 710 715 720

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

725 730 735

Ser Leu Gln Pro Glu Asp Ser Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr

740 745 750

Gly Tyr Leu His Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg

755 760 765

<210> 139

<211> 2304

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - polynucleotide construct for 5T4 x 4-1BB

<400> 139

atggaagcac cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccggt 60

gaggtgcagc tgttggagag cgggggaggc ttggtacagc ctggggggtc cctgcgcctc 120

tcctgtgcag ccagcggatt caccttttct cacggttcta tgtactgggt ccgccaggct 180

ccagggaagg ggctggagtg ggtctcatct atttcttctg gttctggttc tacatactat 240

gcagactccg tgaagggccg gttcaccatc tcccatgaca attccaagaa cacgctgtat 300

ctgcaaatga acagcctgcg tgccgaggac acggctgtat attattgtgc gcgctcttct 360

tactacggtt cttactactc tattgactat tggggccagg gaaccctggt caccgtctcc 420

tcaggtggag gtggctccgg gggtggaggt tccggaggag gcggatcagg tggaggcgga 480

agcgacatcc agatgaccca gtctccatcc tccctgagcg catctgtagg agaccgcgtc 540

accatcactt gccgggcaag tcagagcatt agcagctatt taaattggta tcagcagaaa 600

ccagggaaag cccctaagct cctgatctat gctgcatcca gtttgcaaag tggggtccca 660

tcacgtttca gtggcagtgg aagcgggaca gatttcactc tcaccatcag cagtctacaa 720

cctgaagatt ttgcaactta ttactgtcaa cagtactacg acaacctgcc cacttttggc 780

caggggacca agctggagat caaatcgagt gagcccaaat cttctgacaa aactcacaca 840

tgcccaccgt gcccagcacc tgaagccgcg ggtgcaccgt cagtcttcct cttcccccca 900

aaacccaagg acaccctcat gatctcccgg acccctgagg tcacatgcgt ggtggtggac 960

gtgagccacg aagaccctga ggtcaagttc aactggtacg tggacggcgt ggaggtgcat 1020

aatgccaaga caaagccgcg ggagggagcag tacaacagca cgtaccgtgt ggtcagcgtc 1080

ctcaccgtcc tgcaccagga ctggctgaat ggcaaggaat acaagtgcgc ggtctccaac 1140

aaagccctcc cagcccccat cgagaaaacc atctccaaag ccaaagggca gccccgagaa 1200

ccacaggtgt acaccctgcc cccacccgg gatgagctga ccaagaacca ggtcagcctg 1260

acctgcctgg tcaaaggctt ctatccaagc gacatcgccg tggagtggga gagcaatggg 1320

cagccggaga acaactacaa gaccacgcct cccgtgctgg actccgacgg ctccttcttc 1380

ctctacagca agctcaccgt ggacaagagc aggtggcagc aggggaacgt cttctcatgc 1440

tccgtgatgc atgaggctct gcacaaccac tacacgcaga agagcctctc cctgtctccg 1500

ggttccggag gtggcggttc gggaggtggc gggtcaggag gtgggggatc cccttcagaa 1560

gtgcagctgc tggagtccgg aggaggactg gtgcagcctg gcggaagcct gaggctgagc 1620

tgcgctgcct ccggcttcac attcagcagc tatgctatga gctgggtgag gcaagcccct 1680

ggaaagtgcc tggagtgggt gtccgctatc tccggcagcg gcggaagcac ctactacgct 1740

gactccgtca agggcaggtt caccatcagc cgggacaaca gcaagaacac cctgtacctg 1800

cagatgaata gcctcagggc tgaagacacc gctgtgtact actgcgccag gtactatggc 1860

ggctactact ccgcctggat ggactactgg ggacagggca cactggtgac cgtgtccagc 1920

ggcggaggcg gctccggagg cggtggctcc ggaggaggcg gaagcggagg aggaggctcc 1980

gatattcaga tgacacagtc ccctagctcc ctgtccgcca gcgtgggaga tcgggtgacc 2040

atcacctgca gggccagcca gtccatctcc agcttcttaa actggtacca gcagaagcct 2100

ggaaaggctc ccaagctgct gatctacgcc gcttccagcc tccagagcgg cgtgcctagc 2160

aggttctccg gctccggaag cggaacagac ttcaccctga ccatcagctc cctgcagccc 2220

gaggactccg ctacctacta ctgccagcag acctatggct acctgcacac cttcggctgc 2280

ggcacaaagc tggagatcaa gcgc 2304

<210> 140

<211> 768

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - polypeptide construct to 5T4 x 4-1BB

<400> 140

Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro

1 5 10 15

Asp Thr Thr Gly Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val

20 25 30

Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr

35 40 45

Phe Ser His Gly Ser Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly

50 55 60

Leu Glu Trp Val Ser Ser Ile Ser Ser Gly Ser Gly Ser Thr Tyr Tyr

65 70 75 80

Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser His Asp Asn Ser Lys

85 90 95

Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala

100 105 110

Val Tyr Tyr Cys Ala Arg Ser Ser Tyr Tyr Gly Ser Tyr Tyr Ser Ile

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

145 150 155 160

Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val

165 170 175

Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser

180 185 190

Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu

195 200 205

Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser

210 215 220

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln

225 230 235 240

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Asp Asn Leu

245 250 255

Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Ser Ser Glu Pro

260 265 270

Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

275 280 285

Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

290 295 300

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

305 310 315 320

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

325 330 335

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

340 345 350

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

355 360 365

Leu Asn Gly Lys Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala Leu Pro

370 375 380

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

385 390 395 400

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn

405 410 415

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

420 425 430

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

435 440 445

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

450 455 460

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

465 470 475 480

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

485 490 495

Ser Leu Ser Pro Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

500 505 510

Gly Gly Gly Gly Ser Pro Ser Glu Val Gln Leu Leu Glu Ser Gly Gly

515 520 525

Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser

530 535 540

Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro

545 550 555 560

Gly Lys Cys Leu Glu Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser

565 570 575

Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

580 585 590

Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu

595 600 605

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Gly Gly Tyr Tyr Ser

610 615 620

Ala Trp Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

625 630 635 640

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

645 650 655

Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser

660 665 670

Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser

675 680 685

Ile Ser Ser Phe Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

690 695 700

Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser

705 710 715 720

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

725 730 735

Ser Leu Gln Pro Glu Asp Ser Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr

740 745 750

Gly Tyr Leu His Thr Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys Arg

755 760 765

<210> 141

<211> 2304

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - polynucleotide construct for 5T4 x 4-1BB

<400> 141

atggaagcac cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccggt 60

gaggtgcagc tgttggagag cgggggaggc ttggtacagc ctggggggtc cctgcgcctc 120

tcctgtgcag ccagcggatt cacctttgat tacggttcta tgtactgggt ccgccaggct 180

ccagggaagg ggctggagtg ggtctcatct atttcttctg gttctggttc tacatactat 240

gcagactccg tgaagggccg gttcaccatc tcccacgaca attccaagaa cacgctgtat 300

ctgcaaatga acagcctgcg tgccgaggac acggctgtat attattgtgc gcgctcttct 360

tactacggtt cttactactc tattgactat tggggccagg gaaccctggt caccgtctcc 420

tcaggtggag gtggctccgg gggtggaggt tccggaggag gcggatcagg tggaggcgga 480

agcgacatcc agatgaccca gtctccatcc tccctgagcg catctgtagg agaccgcgtc 540

accatcactt gccgggcaag tcagagcatt agcagctatt taaattggta tcagcagaaa 600

ccagggaaag cccctaagct cctgatctat gctgcatcca gtttgcacag tggggtccca 660

tcacgtttca gtggcagtgg aagcgggaca gatttcactc tcaccatcag cagtctgcaa 720

cctgaagatt ttgcaactta ttactgtcaa cagtactacg acaacctgcc cacttttggc 780

caggggacca agctggagat caaatcctcg gagcccaaat cttctgacaa aactcacaca 840

tgcccaccgt gcccagcacc tgaagccgcg ggtgcaccgt cagtcttcct cttcccccca 900

aaacccaagg acaccctcat gatctcccgg acccctgagg tcacatgcgt ggtggtggac 960

gtgagccacg aagaccctga ggtcaagttc aactggtacg tggacggcgt ggaggtgcat 1020

aatgccaaga caaagccgcg ggagggagcag tacaacagca cgtaccgtgt ggtcagcgtc 1080

ctcaccgtcc tgcaccagga ctggctgaat ggcaaggaat acaagtgcgc ggtctccaac 1140

aaagccctcc cagcccccat cgagaaaacc atctccaaag ccaaagggca gccccgagaa 1200

ccacaggtgt acaccctgcc cccacccgg gatgagctga ccaagaacca ggtcagcctg 1260

acctgcctgg tcaaaggctt ctatccaagc gacatcgccg tggagtggga gagcaatggg 1320

cagccggaga acaactacaa gaccacgcct cccgtgctgg actccgacgg ctccttcttc 1380

ctctacagca agctcaccgt ggacaagagc aggtggcagc aggggaacgt cttctcatgc 1440

tccgtgatgc atgaggctct gcacaaccac tacacgcaga agagcctctc cctgtctccg 1500

ggttccggag gtggcggttc gggaggtggc gggtcaggag gtgggggatc cccttcagaa 1560

gtgcagctgc tggagtccgg aggaggactg gtgcagcctg gcggaagcct gaggctgagc 1620

tgcgctgcct ccggcttcac attcagcagc tatgctatga gctgggtgag gcaagcccct 1680

ggaaagtgcc tggagtgggt gtccgctatc tccggcagcg gcggaagcac ctactacgct 1740

gactccgtca agggcaggtt caccatcagc cgggacaaca gcaagaacac cctgtacctg 1800

cagatgaata gcctcagggc tgaagacacc gctgtgtact actgcgccag gtactatggc 1860

ggctactact ccgcctggat ggactactgg ggacagggca cactggtgac cgtgtccagc 1920

ggcggaggcg gctccggagg cggtggctcc ggaggaggcg gaagcggagg aggaggctcc 1980

gatattcaga tgacacagtc ccctagctcc ctgtccgcca gcgtgggaga tcgggtgacc 2040

atcacctgca gggccagcca gtccatctcc agcttcttaa actggtacca gcagaagcct 2100

ggaaaggctc ccaagctgct gatctacgcc gcttccagcc tccagagcgg cgtgcctagc 2160

aggttctccg gctccggaag cggaacagac ttcaccctga ccatcagctc cctgcagccc 2220

gaggacttcg ctacctacta ctgccagcag acctatggct acctgcacac cttcggctgc 2280

ggcacaaagc tggagatcaa gagc 2304

<210> 142

<211> 768

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - polypeptide construct to 5T4 x 4-1BB

<400> 142

Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro

1 5 10 15

Asp Thr Thr Gly Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val

20 25 30

Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr

35 40 45

Phe Asp Tyr Gly Ser Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly

50 55 60

Leu Glu Trp Val Ser Ser Ile Ser Ser Gly Ser Gly Ser Thr Tyr Tyr

65 70 75 80

Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser His Asp Asn Ser Lys

85 90 95

Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala

100 105 110

Val Tyr Tyr Cys Ala Arg Ser Ser Tyr Tyr Gly Ser Tyr Tyr Ser Ile

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

145 150 155 160

Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val

165 170 175

Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser

180 185 190

Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu

195 200 205

Ile Tyr Ala Ala Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser

210 215 220

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln

225 230 235 240

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Asp Asn Leu

245 250 255

Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Ser Ser Glu Pro

260 265 270

Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

275 280 285

Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

290 295 300

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

305 310 315 320

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

325 330 335

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

340 345 350

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

355 360 365

Leu Asn Gly Lys Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala Leu Pro

370 375 380

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

385 390 395 400

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn

405 410 415

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

420 425 430

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

435 440 445

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

450 455 460

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

465 470 475 480

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

485 490 495

Ser Leu Ser Pro Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

500 505 510

Gly Gly Gly Gly Ser Pro Ser Glu Val Gln Leu Leu Glu Ser Gly Gly

515 520 525

Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser

530 535 540

Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro

545 550 555 560

Gly Lys Cys Leu Glu Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser

565 570 575

Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

580 585 590

Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu

595 600 605

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Gly Gly Tyr Tyr Ser

610 615 620

Ala Trp Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

625 630 635 640

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

645 650 655

Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser

660 665 670

Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser

675 680 685

Ile Ser Ser Phe Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

690 695 700

Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser

705 710 715 720

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

725 730 735

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr

740 745 750

Gly Tyr Leu His Thr Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys Ser

755 760 765

<210> 143

<211> 2304

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - polynucleotide construct for 5T4 x 4-1BB

<400> 143

atggaagcac cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccggt 60

gaggtgcagc tgttggagag cgggggaggc ttggtacagc ctggggggtc cctgcgcctc 120

tcctgtgcag ccagcggatt caccttttct tacggttcta tgtactgggt ccgccaggct 180

ccagggaagg ggctggagtg ggtctcatct atttcttctg gttctggttc tacatactat 240

gcagactccg tgaagggccg gttcaccatc tcccatgaca attccaagaa cacgctgtat 300

ctgcaaatga acagcctgcg tgccgaggac acggctgtat attattgtgc gcgctcttct 360

tactacggtt cttactactc tattgactat tggggccagg gaaccctggt caccgtctcc 420

tcaggtggag gtggctccgg gggtggaggt tccggaggag gcggatcagg tggaggcgga 480

agcgacatcc agatgaccca gtctccatcc tccctgagcg catctgtagg agaccgcgtc 540

accatcactt gccgggcaag tcagagcatt agcagctatt taaattggta tcagcagaaa 600

ccagggaaag cccctaagct cctgatctat gctgcatcca gtttgcaaag tggggtccca 660

tcacgtttca gtggcagtgg aagcgggaca gatttcactc tcaccatcag cagtctacaa 720

cctgaagatt ttgcaactta ttactgtcaa cagtactacg acaacctgcc cacttttggc 780

caggggacca agctggagat caaatcctcg gagcccaaat cttctgacaa aactcacaca 840

tgcccaccgt gcccagcacc tgaagccgcg ggtgcaccgt cagtcttcct cttcccccca 900

aaacccaagg acaccctcat gatctcccgg acccctgagg tcacatgcgt ggtggtggac 960

gtgagccacg aagaccctga ggtcaagttc aactggtacg tggacggcgt ggaggtgcat 1020

aatgccaaga caaagccgcg ggagggagcag tacaacagca cgtaccgtgt ggtcagcgtc 1080

ctcaccgtcc tgcaccagga ctggctgaat ggcaaggaat acaagtgcgc ggtctccaac 1140

aaagccctcc cagcccccat cgagaaaacc atctccaaag ccaaagggca gccccgagaa 1200

ccacaggtgt acaccctgcc cccacccgg gatgagctga ccaagaacca ggtcagcctg 1260

acctgcctgg tcaaaggctt ctatccaagc gacatcgccg tggagtggga gagcaatggg 1320

cagccggaga acaactacaa gaccacgcct cccgtgctgg actccgacgg ctccttcttc 1380

ctctacagca agctcaccgt ggacaagagc aggtggcagc aggggaacgt cttctcatgc 1440

tccgtgatgc atgaggctct gcacaaccac tacacgcaga agagcctctc cctgtctccg 1500

ggttccggag gtggcggttc gggaggtggc gggtcaggag gtgggggatc cccttcagaa 1560

gtgcagctgc tggagtccgg aggaggactg gtgcagcctg gcggaagcct gaggctgagc 1620

tgcgctgcct ccggcttcga cttcgagagc tatgctatga gctgggtgag gcaagcccct 1680

ggaaagtgcc tggagtgggt gtccgctatc tccggcagcg gcggaagcac ctactacgct 1740

gactccgtca agggcaggtt caccatcagc cgggacaaca gcaagaacac cctgtacctg 1800

cagatgaata gcctcagggc tgaagacacc gctgtgtact actgcgccag gtactatggc 1860

ggctactact ccgcctggat ggactactgg ggacagggca cactggtgac cgtgtccagc 1920

ggcggaggcg gctccggagg cggtggctcc ggaggaggcg gaagcggagg aggaggctcc 1980

gatattcaga tgacacagtc ccctagctcc ctgtccgcca gcgtgggaga tcgggtgacc 2040

atcacctgca gggccagcca gtccatcagg agcgccctga actggtacca gcagaagcct 2100

ggaaaggctc ccaagctgct gatctacgcc gcttccagcc tccagagcgg cgtgcctagc 2160

aggttctccg gctccggaag cggaacagac ttcaccctga ccatcagctc cctgcagccc 2220

gaggacttcg ctacctacta ctgccagcag acctatggct acctgcacac cttcggctgc 2280

ggcacaaagc tggagatcaa gcgc 2304

<210> 144

<211> 768

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - polypeptide construct to 5T4 x 4-1BB

<400> 144

Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro

1 5 10 15

Asp Thr Thr Gly Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val

20 25 30

Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr

35 40 45

Phe Ser Tyr Gly Ser Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly

50 55 60

Leu Glu Trp Val Ser Ser Ile Ser Ser Gly Ser Gly Ser Thr Tyr Tyr

65 70 75 80

Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser His Asp Asn Ser Lys

85 90 95

Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala

100 105 110

Val Tyr Tyr Cys Ala Arg Ser Ser Tyr Tyr Gly Ser Tyr Tyr Ser Ile

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

145 150 155 160

Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val

165 170 175

Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser

180 185 190

Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu

195 200 205

Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser

210 215 220

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln

225 230 235 240

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Asp Asn Leu

245 250 255

Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Ser Ser Glu Pro

260 265 270

Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

275 280 285

Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

290 295 300

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

305 310 315 320

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

325 330 335

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

340 345 350

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

355 360 365

Leu Asn Gly Lys Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala Leu Pro

370 375 380

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

385 390 395 400

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn

405 410 415

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

420 425 430

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

435 440 445

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

450 455 460

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

465 470 475 480

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

485 490 495

Ser Leu Ser Pro Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

500 505 510

Gly Gly Gly Gly Ser Pro Ser Glu Val Gln Leu Leu Glu Ser Gly Gly

515 520 525

Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser

530 535 540

Gly Phe Asp Phe Glu Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro

545 550 555 560

Gly Lys Cys Leu Glu Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser

565 570 575

Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

580 585 590

Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu

595 600 605

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Gly Gly Tyr Tyr Ser

610 615 620

Ala Trp Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

625 630 635 640

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

645 650 655

Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser

660 665 670

Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser

675 680 685

Ile Arg Ser Ala Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

690 695 700

Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser

705 710 715 720

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

725 730 735

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr

740 745 750

Gly Tyr Leu His Thr Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys Arg

755 760 765

<210> 145

<211> 2304

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - polynucleotide construct for 5T4 x 4-1BB

<400> 145

atggaagcac cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccggt 60

gaggtgcagc tgttggagag cgggggaggc ttggtacagc ctggggggtc cctgcgcctc 120

tcctgtgcag ccagcggatt caccttttct cacggttcta tgtactgggt ccgccaggct 180

ccagggaagg ggctggagtg ggtctcatct atttcttctg gttctggttc tacatactat 240

gcagactccg tgaagggccg gttcaccatc tcccatgaca attccaagaa cacgctgtat 300

ctgcaaatga acagcctgcg tgccgaggac acggctgtat attattgtgc gcgctcttct 360

tactacggtt cttactactc tattgactat tggggccagg gaaccctggt caccgtctcc 420

tcaggtggag gtggctccgg gggtggaggt tccggaggag gcggatcagg tggaggcgga 480

agcgacatcc agatgaccca gtctccatcc tccctgagcg catctgtagg agaccgcgtc 540

accatcactt gccgggcaag tcagagcatt agcagctatt taaattggta tcagcagaaa 600

ccagggaaag cccctaagct cctgatctat gctgcatcca gtttgcaaag tggggtccca 660

tcacgtttca gtggcagtgg aagcgggaca gatttcactc tcaccatcag cagtctacaa 720

cctgaagatt ttgcaactta ttactgtcaa cagtactacg acaacctgcc cacttttggc 780

caggggacca agctggagat caaatcgagt gagcccaaat cttctgacaa aactcacaca 840

tgcccaccgt gcccagcacc tgaagccgcg ggtgcaccgt cagtcttcct cttcccccca 900

aaacccaagg acaccctcat gatctcccgg acccctgagg tcacatgcgt ggtggtggac 960

gtgagccacg aagaccctga ggtcaagttc aactggtacg tggacggcgt ggaggtgcat 1020

aatgccaaga caaagccgcg ggagggagcag tacaacagca cgtaccgtgt ggtcagcgtc 1080

ctcaccgtcc tgcaccagga ctggctgaat ggcaaggaat acaagtgcgc ggtctccaac 1140

aaagccctcc cagcccccat cgagaaaacc atctccaaag ccaaagggca gccccgagaa 1200

ccacaggtgt acaccctgcc cccacccgg gatgagctga ccaagaacca ggtcagcctg 1260

acctgcctgg tcaaaggctt ctatccaagc gacatcgccg tggagtggga gagcaatggg 1320

cagccggaga acaactacaa gaccacgcct cccgtgctgg actccgacgg ctccttcttc 1380

ctctacagca agctcaccgt ggacaagagc aggtggcagc aggggaacgt cttctcatgc 1440

tccgtgatgc atgaggctct gcacaaccac tacacgcaga agagcctctc cctgtctccg 1500

ggttccggag gtggcggttc gggaggtggc gggtcaggag gtgggggatc cccttcagaa 1560

gtgcagctgc tggagtccgg aggaggactg gtgcagcctg gcggaagcct gaggctgagc 1620

tgcgctgcct ccggcttcga cttcgagagc tatgctatga gctgggtgag gcaagcccct 1680

ggaaagtgcc tggagtgggt gtccgctatc tccggcagcg gcggaagcac ctactacgct 1740

gactccgtca agggcaggtt caccatcagc cgggacaaca gcaagaacac cctgtacctg 1800

cagatgaata gcctcagggc tgaagacacc gctgtgtact actgcgccag gtactatggc 1860

ggctactact ccgcctggat ggactactgg ggacagggca cactggtgac cgtgtccagc 1920

ggcggaggcg gctccggagg cggtggctcc ggaggaggcg gaagcggagg aggaggctcc 1980

gatattcaga tgacacagtc ccctagctcc ctgtccgcca gcgtgggaga tcgggtgacc 2040

atcacctgca gggccagcca gtccatcagg agcgccctga actggtacca gcagaagcct 2100

ggaaaggctc ccaagctgct gatctacgcc gcttccagcc tccagagcgg cgtgcctagc 2160

aggttctccg gctccggaag cggaacagac ttcaccctga ccatcagctc cctgcagccc 2220

gaggacttcg ctacctacta ctgccagcag acctatggct acctgcacac cttcggctgc 2280

ggcacaaagc tggagatcaa gcgc 2304

<210> 146

<211> 768

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - polypeptide construct to 5T4 x 4-1BB

<400> 146

Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro

1 5 10 15

Asp Thr Thr Gly Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val

20 25 30

Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr

35 40 45

Phe Ser His Gly Ser Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly

50 55 60

Leu Glu Trp Val Ser Ser Ile Ser Ser Gly Ser Gly Ser Thr Tyr Tyr

65 70 75 80

Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser His Asp Asn Ser Lys

85 90 95

Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala

100 105 110

Val Tyr Tyr Cys Ala Arg Ser Ser Tyr Tyr Gly Ser Tyr Tyr Ser Ile

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

145 150 155 160

Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val

165 170 175

Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser

180 185 190

Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu

195 200 205

Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser

210 215 220

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln

225 230 235 240

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Asp Asn Leu

245 250 255

Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Ser Ser Glu Pro

260 265 270

Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

275 280 285

Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

290 295 300

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

305 310 315 320

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

325 330 335

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

340 345 350

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

355 360 365

Leu Asn Gly Lys Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala Leu Pro

370 375 380

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

385 390 395 400

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn

405 410 415

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

420 425 430

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

435 440 445

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

450 455 460

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

465 470 475 480

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

485 490 495

Ser Leu Ser Pro Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

500 505 510

Gly Gly Gly Gly Ser Pro Ser Glu Val Gln Leu Leu Glu Ser Gly Gly

515 520 525

Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser

530 535 540

Gly Phe Asp Phe Glu Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro

545 550 555 560

Gly Lys Cys Leu Glu Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser

565 570 575

Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

580 585 590

Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu

595 600 605

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Gly Gly Tyr Tyr Ser

610 615 620

Ala Trp Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

625 630 635 640

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

645 650 655

Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser

660 665 670

Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser

675 680 685

Ile Arg Ser Ala Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

690 695 700

Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser

705 710 715 720

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

725 730 735

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr

740 745 750

Gly Tyr Leu His Thr Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys Arg

755 760 765

<210> 147

<211> 2304

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - polynucleotide construct for 5T4 x 4-1BB

<400> 147

atggaagcac cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccggt 60

gaggtgcagc tgttggagag cgggggaggc ttggtacagc ctggggggtc cctgcgcctc 120

tcctgtgcag ccagcggatt caccttttct cacggttcta tgtactgggt ccgccaggct 180

ccagggaagg ggctggagtg ggtctcatct atttcttctg gttctggttc tacatactat 240

gcagactccg tgaagggccg gttcaccatc tcccatgaca attccaagaa cacgctgtat 300

ctgcaaatga acagcctgcg tgccgaggac acggctgtat attattgtgc gcgctcttct 360

tactacggtt cttactactc tattgactat tggggccagg gaaccctggt caccgtctcc 420

tcaggtggag gtggctccgg gggtggaggt tccggaggag gcggatcagg tggaggcgga 480

agcgacatcc agatgaccca gtctccatcc tccctgagcg catctgtagg agaccgcgtc 540

accatcactt gccgggcaag tcagagcatt agcagctatt taaattggta tcagcagaaa 600

ccagggaaag cccctaagct cctgatctat gctgcatcca gtttgcaaag tggggtccca 660

tcacgtttca gtggcagtgg aagcgggaca gatttcactc tcaccatcag cagtctacaa 720

cctgaagatt ttgcaactta ttactgtcaa cagtactacg acaacctgcc cacttttggc 780

caggggacca agctggagat caaatcgagt gagcccaaat cttctgacaa aactcacaca 840

tgcccaccgt gcccagcacc tgaagccgcg ggtgcaccgt cagtcttcct cttcccccca 900

aaacccaagg acaccctcat gatctcccgg acccctgagg tcacatgcgt ggtggtggac 960

gtgagccacg aagaccctga ggtcaagttc aactggtacg tggacggcgt ggaggtgcat 1020

aatgccaaga caaagccgcg ggagggagcag tacaacagca cgtaccgtgt ggtcagcgtc 1080

ctcaccgtcc tgcaccagga ctggctgaat ggcaaggaat acaagtgcgc ggtctccaac 1140

aaagccctcc cagcccccat cgagaaaacc atctccaaag ccaaagggca gccccgagaa 1200

ccacaggtgt acaccctgcc cccacccgg gatgagctga ccaagaacca ggtcagcctg 1260

acctgcctgg tcaaaggctt ctatccaagc gacatcgccg tggagtggga gagcaatggg 1320

cagccggaga acaactacaa gaccacgcct cccgtgctgg actccgacgg ctccttcttc 1380

ctctacagca agctcaccgt ggacaagagc aggtggcagc aggggaacgt cttctcatgc 1440

tccgtgatgc atgaggctct gcacaaccac tacacgcaga agagcctctc cctgtctccg 1500

ggttccggag gtggcggttc gggaggtggc gggtcaggag gtgggggatc cccttcagaa 1560

gtgcagctgc tggagtccgg aggaggactg gtgcagcctg gcggaagcct gaggctgagc 1620

tgcgctgcct ccggcttcga cttcgacagc tatgctatga gctgggtgag gcaagcccct 1680

ggaaagtgcc tggagtgggt gtccgctatc tccggcaggg gcggaagcac ctactacgct 1740

gactccgtca agggcaggtt caccatcagc cgggacaaca gcaagaacac cctgtacctg 1800

cagatgaata gcctcagggc tgaagacacc gctgtgtact actgcgccag gtactatggc 1860

ggctactact ccgcctggat ggactactgg ggacagggca cactggtgac cgtgtccagc 1920

ggcggaggcg gctccggagg cggtggctcc ggaggaggcg gaagcggagg aggaggctcc 1980

gatattcaga tgacacagtc ccctagctcc ctgtccgcca gcgtgggaga tcgggtgacc 2040

atcacctgca gggccagcca gtccatcagg agcgccctga actggtacca gcagaagcct 2100

ggaaaggctc ccaagctgct gatctacgcc gcttccagcc tccagagcgg cgtgcctagc 2160

aggttctccg gctccggaag cggaacagac ttcaccctga ccatcagctc cctgcagccc 2220

gaggacttcg ctacctacta ctgccagcag acctatggct acctgcacac cttcggctgc 2280

ggcacaaagc tggagatcaa gcgc 2304

<210> 148

<211> 768

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - polypeptide construct to 5T4 x 4-1BB

<400> 148

Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro

1 5 10 15

Asp Thr Thr Gly Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val

20 25 30

Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr

35 40 45

Phe Ser His Gly Ser Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly

50 55 60

Leu Glu Trp Val Ser Ser Ile Ser Ser Gly Ser Gly Ser Thr Tyr Tyr

65 70 75 80

Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser His Asp Asn Ser Lys

85 90 95

Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala

100 105 110

Val Tyr Tyr Cys Ala Arg Ser Ser Tyr Tyr Gly Ser Tyr Tyr Ser Ile

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

145 150 155 160

Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val

165 170 175

Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser

180 185 190

Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu

195 200 205

Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser

210 215 220

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln

225 230 235 240

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Asp Asn Leu

245 250 255

Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Ser Ser Glu Pro

260 265 270

Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

275 280 285

Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

290 295 300

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

305 310 315 320

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

325 330 335

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

340 345 350

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

355 360 365

Leu Asn Gly Lys Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala Leu Pro

370 375 380

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

385 390 395 400

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn

405 410 415

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

420 425 430

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

435 440 445

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

450 455 460

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

465 470 475 480

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

485 490 495

Ser Leu Ser Pro Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

500 505 510

Gly Gly Gly Gly Ser Pro Ser Glu Val Gln Leu Leu Glu Ser Gly Gly

515 520 525

Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser

530 535 540

Gly Phe Asp Phe Asp Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro

545 550 555 560

Gly Lys Cys Leu Glu Trp Val Ser Ala Ile Ser Gly Arg Gly Gly Ser

565 570 575

Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

580 585 590

Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu

595 600 605

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Gly Gly Tyr Tyr Ser

610 615 620

Ala Trp Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

625 630 635 640

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

645 650 655

Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser

660 665 670

Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser

675 680 685

Ile Arg Ser Ala Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

690 695 700

Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser

705 710 715 720

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

725 730 735

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr

740 745 750

Gly Tyr Leu His Thr Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys Arg

755 760 765

<210> 149

<211> 2307

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - polynucleotide construct for 5T4 x 4-1BB

<400> 149

atggaagcac cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccggt 60

gaggtgcagc tgttggagag cgggggaggc ttggtacagc ctggggggtc cctgcgcctc 120

tcctgtgcag ccagcggatt caccttttct tacggttcta tgtactgggt ccgccaggct 180

ccagggaagg ggctggagtg ggtctcatct atttcttctg gttctggttc tacatactat 240

gcagactccg tgaagggccg gttcaccatc tcccgtgaca attccaagaa cacgctgtat 300

ctgcaaatga acagcctgcg tgccgaggac acggctgtat attattgtgc gcgctcttct 360

tactacggtt cttactactc tattgactat tggggccagg gaaccctggt caccgtctcc 420

tcaggcggcg gcggcagcgg cggcggcggc agcggcggcg gaggctccgg cggcggcggc 480

agcgacatcc agatgaccca gtctccatcc tccctgagcg catctgtagg agaccgcgtc 540

accatcactt gccgggcaag tcagagcatt agcagctatt taaattggta tcagcagaaa 600

ccagggaaag cccctaagct cctgatctat gctgcatcca gtttgcaaag tggggtccca 660

tcacgtttca gtggcagtgg aagcgggaca gatttcactc tcaccatcag cagtctgcaa 720

cctgaagatt ttgcaactta ttactgtcaa cagtactacg acaacctgcc cacttttggc 780

caggggacca agctggagat caaatcctcg agtgagccca aatcttctga caaaactcac 840

acatgcccac cgtgcccagc acctgaagcc gcgggtgcac cgtcagtctt cctcttcccc 900

ccaaaaccca aggacaccct catgatctcc cggacccctg aggtcacatg cgtggtggtg 960

gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt acgtggacgg cgtggaggtg 1020

cataatgcca agacaaagcc gcgggaggag cagtacaaca gcacgtaccg tgtggtcagc 1080

gtcctcaccg tcctgcacca ggactggctg aatggcaagg aatacaagtg cgcggtctcc 1140

aacaaagccc tcccagcccc catcgagaaa accatctcca aagccaaagg gcagccccga 1200

gaaccacagg tgtacaccct gcccccatcc cgggatgagc tgaccaagaa ccaggtcagc 1260

ctgacctgcc tggtcaaagg cttctatcca agcgacatcg ccgtggagtg ggagagcaat 1320

gggcagccgg agaacaacta caagaccacg cctcccgtgc tggactccga cggctccttc 1380

ttcctctaca gcaagctcac cgtggacaag agcaggtggc agcaggggaa cgtcttctca 1440

tgctccgtga tgcatgaggc tctgcacaac cactacacgc agaagagcct ctccctgtct 1500

ccgggttccg gaggtggcgg ttcgggaggt ggcgggtcag gaggtggggg atccccttca 1560

gaggtgcagc tgttggagag cgggggaggc ttggtacagc ctggggggtc cctgcgcctc 1620

tcctgtgcag ccagcggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 1680

ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactat 1740

gcagactccg tgaagggccg gttcaccatc tcccgtgaca attccaagaa cacgctgtat 1800

ctgcaaatga acagcctgcg tgccgaggac acggctgtat attattgtgc gcgctactac 1860

ggtggttact actctgcttg gatggactat tggggccagg gaaccctggt caccgtctcc 1920

tcaggcggtg gaggcagcgg tggggggtggg tctggaggcg gtggcagtgg cggcggaggc 1980

tctgacatcc agatgaccca gtctccatcc tccctgagcg catctgtagg agaccgcgtc 2040

accatcactt gccggggcaag tcagagcatt agcagctatt taaattggta tcagcagaaa 2100

ccagggaaag cccctaagct cctgatctat gctgcatcca gtttgcaaag tggggtccca 2160

tcacgtttca gtggcagtgg aagcgggaca gatttcactc tcaccatcag cagtctgcaa 2220

cctgaagatt ttgcaactta ttactgtcaa cagacttacg gttacctgca cacttttggc 2280

caggggacca agctggagat caaatca 2307

<210> 150

<211> 769

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - polypeptide construct to 5T4 x 4-1BB

<400> 150

Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro

1 5 10 15

Asp Thr Thr Gly Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val

20 25 30

Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr

35 40 45

Phe Ser Tyr Gly Ser Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly

50 55 60

Leu Glu Trp Val Ser Ser Ile Ser Ser Gly Ser Gly Ser Thr Tyr Tyr

65 70 75 80

Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys

85 90 95

Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala

100 105 110

Val Tyr Tyr Cys Ala Arg Ser Ser Tyr Tyr Gly Ser Tyr Tyr Ser Ile

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

145 150 155 160

Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val

165 170 175

Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser

180 185 190

Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu

195 200 205

Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser

210 215 220

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln

225 230 235 240

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Asp Asn Leu

245 250 255

Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Ser Ser Ser Glu

260 265 270

Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

275 280 285

Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

290 295 300

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

305 310 315 320

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

325 330 335

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

340 345 350

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

355 360 365

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala Leu

370 375 380

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

385 390 395 400

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys

405 410 415

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

420 425 430

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

435 440 445

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

450 455 460

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

465 470 475 480

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

485 490 495

Leu Ser Leu Ser Pro Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

500 505 510

Ser Gly Gly Gly Gly Ser Pro Ser Glu Val Gln Leu Leu Glu Ser Gly

515 520 525

Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala

530 535 540

Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala

545 550 555 560

Pro Gly Lys Gly Leu Glu Trp Val Ser Ala Ile Ser Gly Ser Gly Gly

565 570 575

Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg

580 585 590

Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala

595 600 605

Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Gly Gly Tyr Tyr

610 615 620

Ser Ala Trp Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

625 630 635 640

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

645 650 655

Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu

660 665 670

Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln

675 680 685

Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala

690 695 700

Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro

705 710 715 720

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

725 730 735

Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr

740 745 750

Tyr Gly Tyr Leu His Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

755 760 765

Ser

<210> 151

<211> 2304

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - polynucleotide construct for 5T4 x 4-1BB

<400> 151

atggaagcac cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccggt 60

gaggtgcagc tgttggagag cgggggaggc ttggtacagc ctggggggtc cctgcgcctc 120

tcctgtgcag ccagcggatt caccttttct tacggttcta tgtactgggt ccgccaggct 180

ccagggaagg ggctggagtg ggtctcatct atttcttctg gttctggttc tacatactat 240

gcagactccg tgaagggccg gttcaccatc tcccgtgaca attccaagaa cacgctgtat 300

ctgcaaatga acagcctgcg tgccgaggac acggctgtat attattgtgc gcgctcttct 360

tactacggtt cttactactc tattgactat tggggccagg gaaccctggt caccgtctcc 420

tcaggcggcg gcggcagcgg cggcggcggc agcggcggcg gaggctccgg cggcggcggc 480

agcgacatcc agatgaccca gtctccatcc tccctgagcg catctgtagg agaccgcgtc 540

accatcactt gccgggcaag tcagagcatt agcagctatt taaattggta tcagcagaaa 600

ccagggaaag cccctaagct cctgatctat gctgcatcca gtttgcaaag tggggtccca 660

tcacgtttca gtggcagtgg aagcgggaca gatttcactc tcaccatcag cagtctgcaa 720

cctgaagatt ttgcaactta ttactgtcaa cagtactacg acaacctgcc cacttttggc 780

caggggacca agctggagat caaatcctcg agtgagccca aatcttctga caaaactcac 840

acatgcccac cgtgcccagc acctgaagcc gcgggtgcac cgtcagtctt cctcttcccc 900

ccaaaaccca aggacaccct catgatctcc cggacccctg aggtcacatg cgtggtggtg 960

gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt acgtggacgg cgtggaggtg 1020

cataatgcca agacaaagcc gcgggaggag cagtacaaca gcacgtaccg tgtggtcagc 1080

gtcctcaccg tcctgcacca ggactggctg aatggcaagg aatacaagtg cgcggtctcc 1140

aacaaagccc tcccagcccc catcgagaaa accatctcca aagccaaagg gcagccccga 1200

gaaccacagg tgtacaccct gcccccatcc cgggatgagc tgaccaagaa ccaggtcagc 1260

ctgacctgcc tggtcaaagg cttctatcca agcgacatcg ccgtggagtg ggagagcaat 1320

gggcagccgg agaacaacta caagaccacg cctcccgtgc tggactccga cggctccttc 1380

ttcctctaca gcaagctcac cgtggacaag agcaggtggc agcaggggaa cgtcttctca 1440

tgctccgtga tgcatgaggc tctgcacaac cactacacgc agaagagcct ctccctgtct 1500

ccgggttccg gaggtggcgg ttcgggaggt ggcgggtcag gaggtggggg atccccttca 1560

gacatccaga tgacccagtc tccatcctcc ctgagcgcat ctgtaggaga ccgcgtcacc 1620

atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 1680

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 1740

cgtttcagtg gcagtggaag cgggacagat ttcactctca ccatcagcag tctgcaacct 1800

gaagattttg caacttatta ctgtcaacag acttacggtt acctgcacac ttttggccag 1860

gggaccaagc tggagatcaa aggcggtgga ggcagcggtg ggggtgggtc tggaggcggt 1920

ggcagtggcg gcggaggctc tgaggtgcag ctgttggaga gcgggggagg cttggtacag 1980

cctggggggt ccctgcgcct ctcctgtgca gccagcggat tcacctttag cagctatgcc 2040

atgagctggg tccgccaggc tccagggaag gggctggagt gggtctcagc tattagtggt 2100

agtggtggta gcacatacta tgcagactcc gtgaagggcc ggttcaccat ctcccgtgac 2160

aattccaaga acacgctgta tctgcaaatg aacagcctgc gtgccgagga cacggctgta 2220

tattattgtg cgcgctacta cggtggttac tactctgctt ggatggacta ttggggccag 2280

ggaaccctgg tcaccgtctc ctca 2304

<210> 152

<211> 768

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - polypeptide construct to 5T4 x 4-1BB

<400> 152

Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro

1 5 10 15

Asp Thr Thr Gly Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val

20 25 30

Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr

35 40 45

Phe Ser Tyr Gly Ser Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly

50 55 60

Leu Glu Trp Val Ser Ser Ile Ser Ser Gly Ser Gly Ser Thr Tyr Tyr

65 70 75 80

Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys

85 90 95

Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala

100 105 110

Val Tyr Tyr Cys Ala Arg Ser Ser Tyr Tyr Gly Ser Tyr Tyr Ser Ile

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

145 150 155 160

Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val

165 170 175

Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser

180 185 190

Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu

195 200 205

Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser

210 215 220

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln

225 230 235 240

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Asp Asn Leu

245 250 255

Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Ser Ser Ser Glu

260 265 270

Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

275 280 285

Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

290 295 300

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

305 310 315 320

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

325 330 335

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

340 345 350

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

355 360 365

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala Leu

370 375 380

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

385 390 395 400

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys

405 410 415

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

420 425 430

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

435 440 445

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

450 455 460

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

465 470 475 480

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

485 490 495

Leu Ser Leu Ser Pro Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

500 505 510

Ser Gly Gly Gly Gly Ser Pro Ser Asp Ile Gln Met Thr Gln Ser Pro

515 520 525

Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg

530 535 540

Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro

545 550 555 560

Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser

565 570 575

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

580 585 590

Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

595 600 605

Gln Gln Thr Tyr Gly Tyr Leu His Thr Phe Gly Gln Gly Thr Lys Leu

610 615 620

Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

625 630 635 640

Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Ser Gly Gly

645 650 655

Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser

660 665 670

Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro

675 680 685

Gly Lys Gly Leu Glu Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser

690 695 700

Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

705 710 715 720

Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu

725 730 735

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Gly Gly Tyr Tyr Ser

740 745 750

Ala Trp Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

755 760 765

<210> 153

<211> 2307

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - polynucleotide construct for 5T4 x 4-1BB

<400> 153

atggaagcac cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccggt 60

gaggtgcagc tgttggagag cgggggaggc ttggtacagc ctggggggtc cctgcgcctc 120

tcctgtgcag ccagcggatt caccttttct tacggttcta tgtactgggt ccgccaggct 180

ccagggaagg ggctggagtg ggtctcatct atttcttctg gttctggttc tacatactat 240

gcagactccg tgaagggccg gttcaccatc tcccgtgaca attccaagaa cacgctgtat 300

ctgcaaatga acagcctgcg tgccgaggac acggctgtat attattgtgc gcgctcttct 360

tactacggtt cttactactc tattgactat tggggccagg gaaccctggt caccgtctcc 420

tcaggcggcg gcggcagcgg cggcggcggc agcggcggcg gaggctccgg cggcggcggc 480

agcgacatcc agatgaccca gtctccatcc tccctgagcg catctgtagg agaccgcgtc 540

accatcactt gccgggcaag tcagagcatt agcagctatt taaattggta tcagcagaaa 600

ccagggaaag cccctaagct cctgatctat gctgcatcca gtttgcaaag tggggtccca 660

tcacgtttca gtggcagtgg aagcgggaca gatttcactc tcaccatcag cagtctgcaa 720

cctgaagatt ttgcaactta ttactgtcaa cagtactacg acaacctgcc cacttttggc 780

caggggacca agctggagat caaatcctcg agtgagccca aatcttctga caaaactcac 840

acatgcccac cgtgcccagc acctgaagcc gcgggtgcac cgtcagtctt cctcttcccc 900

ccaaaaccca aggacaccct catgatctcc cggacccctg aggtcacatg cgtggtggtg 960

gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt acgtggacgg cgtggaggtg 1020

cataatgcca agacaaagcc gcgggaggag cagtacaaca gcacgtaccg tgtggtcagc 1080

gtcctcaccg tcctgcacca ggactggctg aatggcaagg aatacaagtg cgcggtctcc 1140

aacaaagccc tcccagcccc catcgagaaa accatctcca aagccaaagg gcagccccga 1200

gaaccacagg tgtacaccct gcccccatcc cgggatgagc tgaccaagaa ccaggtcagc 1260

ctgacctgcc tggtcaaagg cttctatcca agcgacatcg ccgtggagtg ggagagcaat 1320

gggcagccgg agaacaacta caagaccacg cctcccgtgc tggactccga cggctccttc 1380

ttcctctaca gcaagctcac cgtggacaag agcaggtggc agcaggggaa cgtcttctca 1440

tgctccgtga tgcatgaggc tctgcacaac cactacacgc agaagagcct ctccctgtct 1500

ccgggttccg gaggtggcgg ttcgggaggt ggcgggtcag gaggtggggg atccccttca 1560

gaggtgcagc tgttggagag cgggggaggc ttggtacagc ctggggggtc cctgcgcctc 1620

tcctgtgcag ccagcggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 1680

ccagggaagt gcctggagtg ggtctcagct attagtggta gtggtggtag cacatactat 1740

gcagactccg tgaagggccg gttcaccatc tcccgtgaca attccaagaa cacgctgtat 1800

ctgcaaatga acagcctgcg tgccgaggac acggctgtat attattgtgc gcgctactac 1860

ggtggttact actctgcttg gatggactat tggggccagg gaaccctggt caccgtctcc 1920

tcaggcggtg gaggcagcgg tggggggtggg tctggaggcg gtggcagtgg cggcggaggc 1980

tctgacatcc agatgaccca gtctccatcc tccctgagcg catctgtagg agaccgcgtc 2040

accatcactt gccggggcaag tcagagcatt agcagctatt taaattggta tcagcagaaa 2100

ccagggaaag cccctaagct cctgatctat gctgcatcca gtttgcaaag tggggtccca 2160

tcacgtttca gtggcagtgg aagcgggaca gatttcactc tcaccatcag cagtctgcaa 2220

cctgaagatt ttgcaactta ttactgtcaa cagacttacg gttacctgca cacttttggc 2280

tgcgggacca agctggagat caaatca 2307

<210> 154

<211> 769

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - polypeptide construct to 5T4 x 4-1BB

<400> 154

Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro

1 5 10 15

Asp Thr Thr Gly Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val

20 25 30

Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr

35 40 45

Phe Ser Tyr Gly Ser Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly

50 55 60

Leu Glu Trp Val Ser Ser Ile Ser Ser Gly Ser Gly Ser Thr Tyr Tyr

65 70 75 80

Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys

85 90 95

Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala

100 105 110

Val Tyr Tyr Cys Ala Arg Ser Ser Tyr Tyr Gly Ser Tyr Tyr Ser Ile

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

145 150 155 160

Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val

165 170 175

Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser

180 185 190

Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu

195 200 205

Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser

210 215 220

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln

225 230 235 240

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Asp Asn Leu

245 250 255

Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Ser Ser Ser Glu

260 265 270

Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

275 280 285

Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

290 295 300

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

305 310 315 320

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

325 330 335

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

340 345 350

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

355 360 365

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala Leu

370 375 380

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

385 390 395 400

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys

405 410 415

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

420 425 430

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

435 440 445

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

450 455 460

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

465 470 475 480

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

485 490 495

Leu Ser Leu Ser Pro Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

500 505 510

Ser Gly Gly Gly Gly Ser Pro Ser Glu Val Gln Leu Leu Glu Ser Gly

515 520 525

Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala

530 535 540

Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala

545 550 555 560

Pro Gly Lys Cys Leu Glu Trp Val Ser Ala Ile Ser Gly Ser Gly Gly

565 570 575

Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg

580 585 590

Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala

595 600 605

Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Gly Gly Tyr Tyr

610 615 620

Ser Ala Trp Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

625 630 635 640

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

645 650 655

Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu

660 665 670

Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln

675 680 685

Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala

690 695 700

Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro

705 710 715 720

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

725 730 735

Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr

740 745 750

Tyr Gly Tyr Leu His Thr Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys

755 760 765

Ser

<210> 155

<211> 2304

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - polynucleotide construct for 5T4 x 4-1BB

<400> 155

atggaagcac cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccggt 60

gaggtgcagc tgttggagag cgggggaggc ttggtacagc ctggggggtc cctgcgcctc 120

tcctgtgcag ccagcggatt caccttttct tacggttcta tgtactgggt ccgccaggct 180

ccagggaagg ggctggagtg ggtctcatct atttcttctg gttctggttc tacatactat 240

gcagactccg tgaagggccg gttcaccatc tcccgtgaca attccaagaa cacgctgtat 300

ctgcaaatga acagcctgcg tgccgaggac acggctgtat attattgtgc gcgctcttct 360

tactacggtt cttactactc tattgactat tggggccagg gaaccctggt caccgtctcc 420

tcaggcggcg gcggcagcgg cggcggcggc agcggcggcg gaggctccgg cggcggcggc 480

agcgacatcc agatgaccca gtctccatcc tccctgagcg catctgtagg agaccgcgtc 540

accatcactt gccgggcaag tcagagcatt agcagctatt taaattggta tcagcagaaa 600

ccagggaaag cccctaagct cctgatctat gctgcatcca gtttgcaaag tggggtccca 660

tcacgtttca gtggcagtgg aagcgggaca gatttcactc tcaccatcag cagtctgcaa 720

cctgaagatt ttgcaactta ttactgtcaa cagtactacg acaacctgcc cacttttggc 780

caggggacca agctggagat caaatcctcg agtgagccca aatcttctga caaaactcac 840

acatgcccac cgtgcccagc acctgaagcc gcgggtgcac cgtcagtctt cctcttcccc 900

ccaaaaccca aggacaccct catgatctcc cggacccctg aggtcacatg cgtggtggtg 960

gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt acgtggacgg cgtggaggtg 1020

cataatgcca agacaaagcc gcgggaggag cagtacaaca gcacgtaccg tgtggtcagc 1080

gtcctcaccg tcctgcacca ggactggctg aatggcaagg aatacaagtg cgcggtctcc 1140

aacaaagccc tcccagcccc catcgagaaa accatctcca aagccaaagg gcagccccga 1200

gaaccacagg tgtacaccct gcccccatcc cgggatgagc tgaccaagaa ccaggtcagc 1260

ctgacctgcc tggtcaaagg cttctatcca agcgacatcg ccgtggagtg ggagagcaat 1320

gggcagccgg agaacaacta caagaccacg cctcccgtgc tggactccga cggctccttc 1380

ttcctctaca gcaagctcac cgtggacaag agcaggtggc agcaggggaa cgtcttctca 1440

tgctccgtga tgcatgaggc tctgcacaac cactacacgc agaagagcct ctccctgtct 1500

ccgggttccg gaggtggcgg ttcgggaggt ggcgggtcag gaggtggggg atccccttca 1560

gacatccaga tgacccagtc tccatcctcc ctgagcgcat ctgtaggaga ccgcgtcacc 1620

atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 1680

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 1740

cgtttcagtg gcagtggaag cgggacagat ttcactctca ccatcagcag tctgcaacct 1800

gaagattttg caacttatta ctgtcaacag acttacggtt acctgcacac ttttggctgc 1860

gggaccaagc tggagatcaa aggcggtgga ggcagcggtg ggggtgggtc tggaggcggt 1920

ggcagtggcg gcggaggctc tgaggtgcag ctgttggaga gcgggggagg cttggtacag 1980

cctggggggt ccctgcgcct ctcctgtgca gccagcggat tcacctttag cagctatgcc 2040

atgagctggg tccgccaggc tccagggaag tgcctggagt gggtctcagc tattagtggt 2100

agtggtggta gcacatacta tgcagactcc gtgaagggcc ggttcaccat ctcccgtgac 2160

aattccaaga acacgctgta tctgcaaatg aacagcctgc gtgccgagga cacggctgta 2220

tattattgtg cgcgctacta cggtggttac tactctgctt ggatggacta ttggggccag 2280

ggaaccctgg tcaccgtctc ctca 2304

<210> 156

<211> 768

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - polypeptide construct to 5T4 x 4-1BB

<400> 156

Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro

1 5 10 15

Asp Thr Thr Gly Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val

20 25 30

Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr

35 40 45

Phe Ser Tyr Gly Ser Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly

50 55 60

Leu Glu Trp Val Ser Ser Ile Ser Ser Gly Ser Gly Ser Thr Tyr Tyr

65 70 75 80

Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys

85 90 95

Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala

100 105 110

Val Tyr Tyr Cys Ala Arg Ser Ser Tyr Tyr Gly Ser Tyr Tyr Ser Ile

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

145 150 155 160

Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val

165 170 175

Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser

180 185 190

Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu

195 200 205

Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser

210 215 220

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln

225 230 235 240

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Asp Asn Leu

245 250 255

Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Ser Ser Ser Glu

260 265 270

Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

275 280 285

Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

290 295 300

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

305 310 315 320

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

325 330 335

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

340 345 350

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

355 360 365

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala Leu

370 375 380

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

385 390 395 400

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys

405 410 415

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

420 425 430

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

435 440 445

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

450 455 460

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

465 470 475 480

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

485 490 495

Leu Ser Leu Ser Pro Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

500 505 510

Ser Gly Gly Gly Gly Ser Pro Ser Asp Ile Gln Met Thr Gln Ser Pro

515 520 525

Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg

530 535 540

Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro

545 550 555 560

Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser

565 570 575

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

580 585 590

Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

595 600 605

Gln Gln Thr Tyr Gly Tyr Leu His Thr Phe Gly Cys Gly Thr Lys Leu

610 615 620

Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

625 630 635 640

Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Ser Gly Gly

645 650 655

Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser

660 665 670

Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro

675 680 685

Gly Lys Cys Leu Glu Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser

690 695 700

Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

705 710 715 720

Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu

725 730 735

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Gly Gly Tyr Tyr Ser

740 745 750

Ala Trp Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

755 760 765

<210> 157

<211> 699

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragment

<400> 157

tcgagtgagc ccaaatcttc tgacaaaact cacacatgcc caccgtgccc agcacctgaa 60

gccgcgggtg caccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc 120

tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc 180

aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcggggag 240

gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg 300

ctgaatggca aggaatacaa gtgcgcggtc tccaacaaag ccctcccagc ccccatcgag 360

aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca 420

tcccgggatg agctgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat 480

ccaagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc 540

acgcctcccg tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac 600

aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac 660

aaccactaca cgcagaagag cctctccctg tctccggggt 699

<210> 158

<211> 233

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragment

<400> 158

Ser Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys

1 5 10 15

Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro

20 25 30

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

35 40 45

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

50 55 60

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

65 70 75 80

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

85 90 95

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Ala Val Ser Asn

100 105 110

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

115 120 125

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

130 135 140

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

145 150 155 160

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

165 170 175

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

180 185 190

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

195 200 205

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

210 215 220

Gln Lys Ser Leu Ser Leu Ser Pro Gly

225 230

<210> 159

<211> 693

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragment

<400> 159

gagcccaaat cttctgacaa aactcacaca tgcccaccgt gcccagcacc tgaagccgcg 60

ggtgcaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 120

acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 180

aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggagggagcag 240

tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 300

ggcaaggaat acaagtgcgc ggtctccaac aaagccctcc cagcccccat cgagaaaacc 360

atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 420

gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatccaagc 480

gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 540

cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 600

aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 660

tacacgcaga agagcctctc cctgtctccg ggt 693

<210> 160

<211> 231

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - antibody fragment

<400> 160

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly

225 230

<210> 161

<211> 768

<212> DNA

<213> Homo sapiens

<400> 161

atgggaaaca gctgttacaa catagtagcc actctgttgc tggtcctcaa ctttgagagg 60

acaagatcat tgcaggatcc ttgtagtaac tgcccagctg gtacattctg tgataataac 120

aggaatcaga tttgcagtcc ctgtcctcca aatagtttct ccagcgcagg tggacaaagg 180

acctgtgaca tatgcaggca gtgtaaaggt gttttcagga cccaggaagga gtgttcctcc 240

accagcaatg cagagtgtga ctgcactcca gggtttcact gcctgggggc aggatgcagc 300

atgtgtgaac aggattgtaa acaaggtcaa gaactgacaa aaaaaggttg taaagactgt 360

tgctttggga catttaacga tcagaaacgt ggcatctgtc gaccctggac aaactgttct 420

ttggatggaa agtctgtgct tgtgaatggg acgaaggaga gggacgtggt ctgtggacca 480

tctccagccg acctctctcc gggagcatcc tctgtgaccc cgcctgcccc tgcgagagag 540

ccaggacact ctccgcagat catctccttc tttcttgcgc tgacgtcgac tgcgttgctc 600

ttcctgctgt tcttcctcac gctccgtttc tctgttgtta aacggggcag aaagaaactc 660

ctgtatatat tcaaacaacc atttatgaga ccagtacaaa ctactcaaga ggaagatggc 720

tgtagctgcc gatttccaga agaagaagaa ggagatgtg aactgtga 768

<210> 162

<211> 255

<212> PRT

<213> Homo sapiens

<400> 162

Met Gly Asn Ser Cys Tyr Asn Ile Val Ala Thr Leu Leu Leu Val Leu

1 5 10 15

Asn Phe Glu Arg Thr Arg Ser Leu Gln Asp Pro Cys Ser Asn Cys Pro

20 25 30

Ala Gly Thr Phe Cys Asp Asn Asn Arg Asn Gln Ile Cys Ser Pro Cys

35 40 45

Pro Pro Asn Ser Phe Ser Ser Ala Gly Gly Gln Arg Thr Cys Asp Ile

50 55 60

Cys Arg Gln Cys Lys Gly Val Phe Arg Thr Arg Lys Glu Cys Ser Ser

65 70 75 80

Thr Ser Asn Ala Glu Cys Asp Cys Thr Pro Gly Phe His Cys Leu Gly

85 90 95

Ala Gly Cys Ser Met Cys Glu Gln Asp Cys Lys Gln Gly Gln Glu Leu

100 105 110

Thr Lys Lys Gly Cys Lys Asp Cys Cys Phe Gly Thr Phe Asn Asp Gln

115 120 125

Lys Arg Gly Ile Cys Arg Pro Trp Thr Asn Cys Ser Leu Asp Gly Lys

130 135 140

Ser Val Leu Val Asn Gly Thr Lys Glu Arg Asp Val Val Cys Gly Pro

145 150 155 160

Ser Pro Ala Asp Leu Ser Pro Gly Ala Ser Ser Val Thr Pro Pro Ala

165 170 175

Pro Ala Arg Glu Pro Gly His Ser Pro Gln Ile Ile Ser Phe Phe Leu

180 185 190

Ala Leu Thr Ser Thr Ala Leu Leu Phe Leu Leu Phe Phe Leu Thr Leu

195 200 205

Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe

210 215 220

Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly

225 230 235 240

Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

245 250 255

<210> 163

<211> 1263

<212> DNA

<213> Homo sapiens

<400> 163

atgcctgggg ggtgctcccg gggccccgcc gccggggacg ggcgtctgcg gctggcgcga 60

ctagcgctgg tactcctggg ctgggtctcc tcgtcttctc ccacctcctc ggcatcctcc 120

ttctcctcct cggcgccgtt cctggcttcc gccgtgtccg cccagccccc gctgccggac 180

cagtgccccg cgctgtgcga gtgctccgag gcagcgcgca cagtcaagtg cgttaaccgc 240

aatctgaccg aggtgcccac ggacctgccc gcctacgtgc gcaacctctt ccttaccggc 300

aaccagctgg ccgtgctccc tgccggcgcc ttcgcccgcc ggccgccgct ggcggagctg 360

gccgcgctca acctcagcgg cagccgcctg gacgaggtgc gcgcgggcgc cttcgagcat 420

ctgcccagcc tgcgccagct cgacctcagc cacaacccac tggccgacct cagtcccttc 480

gctttctcgg gcagcaatgc cagcgtctcg gcccccagtc cccttgtgga actgatcctg 540

aaccacatcg tgccccctga agatgagcgg cagaaccgga gcttcgaggg catggtggtg 600

gcggccctgc tggcgggccg tgcactgcag gggctccgcc gcttggagct ggccagcaac 660

cacttccttt acctgccgcg ggatgtgctg gcccaactgc ccagcctcag gcacctggac 720

ttaagtaata attcgctggt gagcctgacc tacgtgtcct tccgcaacct gacacatcta 780

gaaagcctcc acctggagga caatgccctc aaggtccttc acaatggcac cctggctgag 840

ttgcaaggtc taccccacat tagggttttc ctggacaaca atccctgggt ctgcgactgc 900

cacatggcag acatggtgac ctggctcaag gaaacagagg tagtgcaggg caaagaccgg 960

ctcacctgtg catatccgga aaaaatgagg aatcgggtcc tcttggaact caacagtgct 1020

gacctggact gtgacccgat tcttccccca tccctgcaaa cctcttatgt cttcctgggt 1080

attgttttag ccctgatagg cgctattttc ctcctggttt tgtatttgaa ccgcaagggg 1140

ataaaaaagt ggatgcataa catcagagat gcctgcaggg atcacatgga agggtatcat 1200

tacagatatg aaatcaatgc ggaccccaga ttaacgaacc tcagttctaa ctcggatgtc 1260

tga 1263

<210> 164

<211> 420

<212> PRT

<213> Homo sapiens

<400> 164

Met Pro Gly Gly Cys Ser Arg Gly Pro Ala Ala Gly Asp Gly Arg Leu

1 5 10 15

Arg Leu Ala Arg Leu Ala Leu Val Leu Leu Gly Trp Val Ser Ser Ser

20 25 30

Ser Pro Thr Ser Ser Ala Ser Ser Phe Ser Ser Ser Ala Pro Phe Leu

35 40 45

Ala Ser Ala Val Ser Ala Gln Pro Pro Leu Pro Asp Gln Cys Pro Ala

50 55 60

Leu Cys Glu Cys Ser Glu Ala Ala Arg Thr Val Lys Cys Val Asn Arg

65 70 75 80

Asn Leu Thr Glu Val Pro Thr Asp Leu Pro Ala Tyr Val Arg Asn Leu

85 90 95

Phe Leu Thr Gly Asn Gln Leu Ala Val Leu Pro Ala Gly Ala Phe Ala

100 105 110

Arg Arg Pro Pro Leu Ala Glu Leu Ala Ala Leu Asn Leu Ser Gly Ser

115 120 125

Arg Leu Asp Glu Val Arg Ala Gly Ala Phe Glu His Leu Pro Ser Leu

130 135 140

Arg Gln Leu Asp Leu Ser His Asn Pro Leu Ala Asp Leu Ser Pro Phe

145 150 155 160

Ala Phe Ser Gly Ser Asn Ala Ser Val Ser Ala Pro Ser Pro Leu Val

165 170 175

Glu Leu Ile Leu Asn His Ile Val Pro Pro Glu Asp Glu Arg Gln Asn

180 185 190

Arg Ser Phe Glu Gly Met Val Val Ala Ala Leu Leu Ala Gly Arg Ala

195 200 205

Leu Gln Gly Leu Arg Arg Leu Glu Leu Ala Ser Asn His Phe Leu Tyr

210 215 220

Leu Pro Arg Asp Val Leu Ala Gln Leu Pro Ser Leu Arg His Leu Asp

225 230 235 240

Leu Ser Asn Asn Ser Leu Val Ser Leu Thr Tyr Val Ser Phe Arg Asn

245 250 255

Leu Thr His Leu Glu Ser Leu His Leu Glu Asp Asn Ala Leu Lys Val

260 265 270

Leu His Asn Gly Thr Leu Ala Glu Leu Gln Gly Leu Pro His Ile Arg

275 280 285

Val Phe Leu Asp Asn Asn Pro Trp Val Cys Asp Cys His Met Ala Asp

290 295 300

Met Val Thr Trp Leu Lys Glu Thr Glu Val Val Gln Gly Lys Asp Arg

305 310 315 320

Leu Thr Cys Ala Tyr Pro Glu Lys Met Arg Asn Arg Val Leu Leu Glu

325 330 335

Leu Asn Ser Ala Asp Leu Asp Cys Asp Pro Ile Leu Pro Pro Ser Leu

340 345 350

Gln Thr Ser Tyr Val Phe Leu Gly Ile Val Leu Ala Leu Ile Gly Ala

355 360 365

Ile Phe Leu Leu Val Leu Tyr Leu Asn Arg Lys Gly Ile Lys Lys Trp

370 375 380

Met His Asn Ile Arg Asp Ala Cys Arg Asp His Met Glu Gly Tyr His

385 390 395 400

Tyr Arg Tyr Glu Ile Asn Ala Asp Pro Arg Leu Thr Asn Leu Ser Ser

405 410 415

Asn Ser Asp Val

420

<210> 165

<211> 558

<212> DNA

<213> Homo sapiens

<400> 165

atgggaaaca gctgttacaa catagtagcc actctgttgc tggtcctcaa ctttgagagg 60

acaagatcat tgcaggatcc ttgtagtaac tgcccagctg gtacattctg tgataataac 120

aggaatcaga tttgcagtcc ctgtcctcca aatagtttct ccagcgcagg tggacaaagg 180

acctgtgaca tatgcaggca gtgtaaaggt gttttcagga cccaggaagga gtgttcctcc 240

accagcaatg cagagtgtga ctgcactcca gggtttcact gcctgggggc aggatgcagc 300

atgtgtgaac aggattgtaa acaaggtcaa gaactgacaa aaaaaggttg taaagactgt 360

tgctttggga catttaacga tcagaaacgt ggcatctgtc gaccctggac aaactgttct 420

ttggatggaa agtctgtgct tgtgaatggg acgaaggaga gggacgtggt ctgtggacca 480

tctccagccg acctctctcc gggagcatcc tctgtgaccc cgcctgcccc tgcgagagag 540

ccaggacact ctccgcag 558

<210> 166

<211> 186

<212> PRT

<213> Homo sapiens

<400> 166

Met Gly Asn Ser Cys Tyr Asn Ile Val Ala Thr Leu Leu Leu Val Leu

1 5 10 15

Asn Phe Glu Arg Thr Arg Ser Leu Gln Asp Pro Cys Ser Asn Cys Pro

20 25 30

Ala Gly Thr Phe Cys Asp Asn Asn Arg Asn Gln Ile Cys Ser Pro Cys

35 40 45

Pro Pro Asn Ser Phe Ser Ser Ala Gly Gly Gln Arg Thr Cys Asp Ile

50 55 60

Cys Arg Gln Cys Lys Gly Val Phe Arg Thr Arg Lys Glu Cys Ser Ser

65 70 75 80

Thr Ser Asn Ala Glu Cys Asp Cys Thr Pro Gly Phe His Cys Leu Gly

85 90 95

Ala Gly Cys Ser Met Cys Glu Gln Asp Cys Lys Gln Gly Gln Glu Leu

100 105 110

Thr Lys Lys Gly Cys Lys Asp Cys Cys Phe Gly Thr Phe Asn Asp Gln

115 120 125

Lys Arg Gly Ile Cys Arg Pro Trp Thr Asn Cys Ser Leu Asp Gly Lys

130 135 140

Ser Val Leu Val Asn Gly Thr Lys Glu Arg Asp Val Val Cys Gly Pro

145 150 155 160

Ser Pro Ala Asp Leu Ser Pro Gly Ala Ser Ser Val Thr Pro Pro Ala

165 170 175

Pro Ala Arg Glu Pro Gly His Ser Pro Gln

180 185

<210> 167

<211> 1065

<212> DNA

<213> Homo sapiens

<400> 167

atgcctgggg ggtgctcccg gggccccgcc gccggggacg ggcgtctgcg gctggcgcga 60

ctagcgctgg tactcctggg ctgggtctcc tcgtcttctc ccacctcctc ggcatcctcc 120

ttctcctcct cggcgccgtt cctggcttcc gccgtgtccg cccagccccc gctgccggac 180

cagtgccccg cgctgtgcga gtgctccgag gcagcgcgca cagtcaagtg cgttaaccgc 240

aatctgaccg aggtgcccac ggacctgccc gcctacgtgc gcaacctctt ccttaccggc 300

aaccagctgg ccgtgctccc tgccggcgcc ttcgcccgcc ggccgccgct ggcggagctg 360

gccgcgctca acctcagcgg cagccgcctg gacgaggtgc gcgcgggcgc cttcgagcat 420

ctgcccagcc tgcgccagct cgacctcagc cacaacccac tggccgacct cagtcccttc 480

gctttctcgg gcagcaatgc cagcgtctcg gcccccagtc cccttgtgga actgatcctg 540

aaccacatcg tgccccctga agatgagcgg cagaaccgga gcttcgaggg catggtggtg 600

gcggccctgc tggcgggccg tgcactgcag gggctccgcc gcttggagct ggccagcaac 660

cacttccttt acctgccgcg ggatgtgctg gcccaactgc ccagcctcag gcacctggac 720

ttaagtaata attcgctggt gagcctgacc tacgtgtcct tccgcaacct gacacatcta 780

gaaagcctcc acctggagga caatgccctc aaggtccttc acaatggcac cctggctgag 840

ttgcaaggtc taccccacat tagggttttc ctggacaaca atccctgggt ctgcgactgc 900

cacatggcag acatggtgac ctggctcaag gaaacagagg tagtgcaggg caaagaccgg 960

ctcacctgtg catatccgga aaaaatgagg aatcgggtcc tcttggaact caacagtgct 1020

gacctggact gtgacccgat tcttccccca tccctgcaaa cctct 1065

<210> 168

<211> 355

<212> PRT

<213> Homo sapiens

<400> 168

Met Pro Gly Gly Cys Ser Arg Gly Pro Ala Ala Gly Asp Gly Arg Leu

1 5 10 15

Arg Leu Ala Arg Leu Ala Leu Val Leu Leu Gly Trp Val Ser Ser Ser

20 25 30

Ser Pro Thr Ser Ser Ala Ser Ser Phe Ser Ser Ser Ala Pro Phe Leu

35 40 45

Ala Ser Ala Val Ser Ala Gln Pro Pro Leu Pro Asp Gln Cys Pro Ala

50 55 60

Leu Cys Glu Cys Ser Glu Ala Ala Arg Thr Val Lys Cys Val Asn Arg

65 70 75 80

Asn Leu Thr Glu Val Pro Thr Asp Leu Pro Ala Tyr Val Arg Asn Leu

85 90 95

Phe Leu Thr Gly Asn Gln Leu Ala Val Leu Pro Ala Gly Ala Phe Ala

100 105 110

Arg Arg Pro Pro Leu Ala Glu Leu Ala Ala Leu Asn Leu Ser Gly Ser

115 120 125

Arg Leu Asp Glu Val Arg Ala Gly Ala Phe Glu His Leu Pro Ser Leu

130 135 140

Arg Gln Leu Asp Leu Ser His Asn Pro Leu Ala Asp Leu Ser Pro Phe

145 150 155 160

Ala Phe Ser Gly Ser Asn Ala Ser Val Ser Ala Pro Ser Pro Leu Val

165 170 175

Glu Leu Ile Leu Asn His Ile Val Pro Pro Glu Asp Glu Arg Gln Asn

180 185 190

Arg Ser Phe Glu Gly Met Val Val Ala Ala Leu Leu Ala Gly Arg Ala

195 200 205

Leu Gln Gly Leu Arg Arg Leu Glu Leu Ala Ser Asn His Phe Leu Tyr

210 215 220

Leu Pro Arg Asp Val Leu Ala Gln Leu Pro Ser Leu Arg His Leu Asp

225 230 235 240

Leu Ser Asn Asn Ser Leu Val Ser Leu Thr Tyr Val Ser Phe Arg Asn

245 250 255

Leu Thr His Leu Glu Ser Leu His Leu Glu Asp Asn Ala Leu Lys Val

260 265 270

Leu His Asn Gly Thr Leu Ala Glu Leu Gln Gly Leu Pro His Ile Arg

275 280 285

Val Phe Leu Asp Asn Asn Pro Trp Val Cys Asp Cys His Met Ala Asp

290 295 300

Met Val Thr Trp Leu Lys Glu Thr Glu Val Val Gln Gly Lys Asp Arg

305 310 315 320

Leu Thr Cys Ala Tyr Pro Glu Lys Met Arg Asn Arg Val Leu Leu Glu

325 330 335

Leu Asn Ser Ala Asp Leu Asp Cys Asp Pro Ile Leu Pro Pro Ser Leu

340 345 350

Gln Thr Ser

355

<210> 169

<211> 744

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - synthesized scFv sequence

<400> 169

gacatccaga tgacccagtc tccatcctcc ctgagcgcat ctgtaggaga ccgcgtcacc 60

atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

cgtttcagtg gcagtggaag cgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttatta ctgtcaacag acttacggtt acctgcacac ttttggccag 300

gggaccaagc tggagatcaa aggcggtgga ggcagcggtg ggggtgggtc tggaggcggt 360

ggcagtggcg gcggaggctc tgaggtgcag ctgttggaga gcgggggagg cttggtacag 420

cctggggggt ccctgcgcct ctcctgtgca gccagcggat tcacctttag cagctatgcc 480

atgagctggg tccgccaggc tccagggaag gggctggagt gggtctcagc tattagtggt 540

agtggtggta gcacatacta tgcagactcc gtgaagggcc ggttcaccat ctcccgtgac 600

aattccaaga acacgctgta tctgcaaatg aacagcctgc gtgccgagga cacggctgta 660

tattattgtg cgcgctacta cggtggttac tactctgctt ggatggacta ttggggccag 720

ggaaccctgg tcaccgtctc ctca 744

<210> 170

<211> 248

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - synthesized scFv sequence

<400> 170

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr Gly Tyr Leu His

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

115 120 125

Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser

130 135 140

Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala

145 150 155 160

Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser

165 170 175

Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys

180 185 190

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu

195 200 205

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

210 215 220

Arg Tyr Tyr Gly Gly Tyr Tyr Ser Ala Trp Met Asp Tyr Trp Gly Gln

225 230 235 240

Gly Thr Leu Val Thr Val Ser Ser

245

<210> 171

<211> 2298

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - polynucleotide construct for 5T4 x 4-1BB

<400> 171

atggaagcac cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccggt 60

gaggtgcagc tgttggagag cgggggaggc ttggtacagc ctggggggtc cctgcgcctc 120

tcctgtgcag ccagcggatt caccttttct cacggttcta tgtactgggt ccgccaggct 180

ccagggaagg ggctggagtg ggtctcatct atttcttctg gttctggttc tacatactat 240

gcagactccg tgaagggccg gttcaccatc tcccatgaca attccaagaa cacgctgtat 300

ctgcaaatga acagcctgcg tgccgaggac acggctgtat attattgtgc gcgctcttct 360

tactacggtt cttactactc tattgactat tggggccagg gaaccctggt caccgtctcc 420

tcaggtggag gtggctccgg gggtggaggt tccggaggag gcggatcagg tggaggcgga 480

agcgacatcc agatgaccca gtctccatcc tccctgagcg catctgtagg agaccgcgtc 540

accatcactt gccgggcaag tcagagcatt agcagctatt taaattggta tcagcagaaa 600

ccagggaaag cccctaagct cctgatctat gctgcatcca gtttgcaaag tggggtccca 660

tcacgtttca gtggcagtgg aagcgggaca gatttcactc tcaccatcag cagtctacaa 720

cctgaagatt ttgcaactta ttactgtcaa cagtactacg acaacctgcc cacttttggc 780

caggggacca agctggagat caaagagccc aaatcttctg acaaaactca cacatgccca 840

ccgtgcccag cacctgaagc cgcgggtgca ccgtcagtct tcctcttccc cccaaaaccc 900

aaggacaccc tcatgatctc ccggacccct gaggtcacat gcgtggtggt ggacgtgagc 960

cacgaagacc ctgaggtcaa gttcaactgg tacgtggacg gcgtggaggt gcataatgcc 1020

aagacaaagc cgcgggagga gcagtacaac agcacgtacc gtgtggtcag cgtcctcacc 1080

gtcctgcacc aggactggct gaatggcaag gaatacaagt gcgcggtctc caacaaagcc 1140

ctcccagccc ccatcgagaa aaccatctcc aaagccaaag ggcagccccg agaaccacag 1200

gtgtacaccc tgcccccatc ccgggatgag ctgaccaaga accaggtcag cctgacctgc 1260

ctggtcaaag gcttctatcc aagcgacatc gccgtggagt ggggagagcaa tgggcagccg 1320

gagaacaact acaagaccac gcctcccgtg ctggactccg acggctcctt cttcctctac 1380

agcaagctca ccgtggacaa gagcaggtgg cagcagggga acgtcttctc atgctccgtg 1440

atgcatgagg ctctgcacaa ccactacacg cagaagagcc tctccctgtc tccgggttcc 1500

ggaggtggcg gttcgggagg tggcgggtca ggaggtgggg gatccccttc agaagtgcag 1560

ctgctggagt ccggaggagg actggtgcag cctggcggaa gcctgaggct gagctgcgct 1620

gcctccggct tcacattcag cagctatgct atgagctggg tgaggcaagc ccctggaaag 1680

ggcctggagt gggtgtccgc tatctccggc agcggcggaa gcacctacta cgctgactcc 1740

gtcaagggca ggttcaccat cagccgggac aacagcaaga acaccctgta cctgcagatg 1800

aatagcctca gggctgaaga caccgctgtg tactactgcg ccaggtacta tggcggctac 1860

tactccgcct ggatggacta ctggggacag ggcacactgg tgaccgtgtc cagcggcgga 1920

ggcggctccg gaggcggtgg ctccggagga ggcggaagcg gaggaggagg ctccgatatt 1980

cagatgacac agtcccctag ctccctgtcc gccagcgtgg gagatcgggt gaccatcacc 2040

tgcagggcca gccagtccat ctccagcttc ttaaactggt accagcagaa gcctggaaag 2100

gctcccaagc tgctgatcta cgccgcttcc agcctccaga gcggcgtgcc tagcaggttc 2160

tccggctccg gaagcggaac agacttcacc ctgaccatca gctccctgca gcccgaggac 2220

tccgctacct actactgcca gcagacctat ggctacctgc acaccttcgg ccagggcaca 2280

aagctggaga tcaagcgc 2298

<210> 172

<211> 766

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - polypeptide construct to 5T4 x 4-1BB

<400> 172

Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro

1 5 10 15

Asp Thr Thr Gly Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val

20 25 30

Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr

35 40 45

Phe Ser His Gly Ser Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly

50 55 60

Leu Glu Trp Val Ser Ser Ile Ser Ser Gly Ser Gly Ser Thr Tyr Tyr

65 70 75 80

Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser His Asp Asn Ser Lys

85 90 95

Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala

100 105 110

Val Tyr Tyr Cys Ala Arg Ser Ser Tyr Tyr Gly Ser Tyr Tyr Ser Ile

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

145 150 155 160

Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val

165 170 175

Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser

180 185 190

Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu

195 200 205

Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser

210 215 220

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln

225 230 235 240

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Asp Asn Leu

245 250 255

Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Glu Pro Lys Ser

260 265 270

Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala

275 280 285

Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

290 295 300

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

305 310 315 320

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

325 330 335

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

340 345 350

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

355 360 365

Gly Lys Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala Leu Pro Ala Pro

370 375 380

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

385 390 395 400

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

405 410 415

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

420 425 430

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

435 440 445

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

450 455 460

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

465 470 475 480

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

485 490 495

Ser Pro Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

500 505 510

Gly Gly Ser Pro Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu

515 520 525

Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe

530 535 540

Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys

545 550 555 560

Gly Leu Glu Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr

565 570 575

Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

580 585 590

Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

595 600 605

Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Gly Gly Tyr Tyr Ser Ala Trp

610 615 620

Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly

625 630 635 640

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

645 650 655

Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser

660 665 670

Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser

675 680 685

Ser Phe Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu

690 695 700

Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe

705 710 715 720

Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu

725 730 735

Gln Pro Glu Asp Ser Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr Gly Tyr

740 745 750

Leu His Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg

755 760 765

<210> 173

<211> 2298

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - polynucleotide construct for 5T4 x 4-1BB

<400> 173

atggaagcac cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccggt 60

gaggtgcagc tgttggagag cgggggaggc ttggtacagc ctggggggtc cctgcgcctc 120

tcctgtgcag ccagcggatt caccttttct cacggttcta tgtactgggt ccgccaggct 180

ccagggaagg ggctggagtg ggtctcatct atttcttctg gttctggttc tacatactat 240

gcagactccg tgaagggccg gttcaccatc tcccatgaca attccaagaa cacgctgtat 300

ctgcaaatga acagcctgcg tgccgaggac acggctgtat attattgtgc gcgctcttct 360

tactacggtt cttactactc tattgactat tggggccagg gaaccctggt caccgtctcc 420

tcaggtggag gtggctccgg gggtggaggt tccggaggag gcggatcagg tggaggcgga 480

agcgacatcc agatgaccca gtctccatcc tccctgagcg catctgtagg agaccgcgtc 540

accatcactt gccgggcaag tcagagcatt agcagctatt taaattggta tcagcagaaa 600

ccagggaaag cccctaagct cctgatctat gctgcatcca gtttgcaaag tggggtccca 660

tcacgtttca gtggcagtgg aagcgggaca gatttcactc tcaccatcag cagtctacaa 720

cctgaagatt ttgcaactta ttactgtcaa cagtactacg acaacctgcc cacttttggc 780

caggggacca agctggagat caaagagccc aaatcttctg acaaaactca cacatgccca 840

ccgtgcccag cacctgaagc cgcgggtgca ccgtcagtct tcctcttccc cccaaaaccc 900

aaggacaccc tcatgatctc ccggacccct gaggtcacat gcgtggtggt ggacgtgagc 960

cacgaagacc ctgaggtcaa gttcaactgg tacgtggacg gcgtggaggt gcataatgcc 1020

aagacaaagc cgcgggagga gcagtacaac agcacgtacc gtgtggtcag cgtcctcacc 1080

gtcctgcacc aggactggct gaatggcaag gaatacaagt gcgcggtctc caacaaagcc 1140

ctcccagccc ccatcgagaa aaccatctcc aaagccaaag ggcagccccg agaaccacag 1200

gtgtacaccc tgcccccatc ccgggatgag ctgaccaaga accaggtcag cctgacctgc 1260

ctggtcaaag gcttctatcc aagcgacatc gccgtggagt ggggagagcaa tgggcagccg 1320

gagaacaact acaagaccac gcctcccgtg ctggactccg acggctcctt cttcctctac 1380

agcaagctca ccgtggacaa gagcaggtgg cagcagggga acgtcttctc atgctccgtg 1440

atgcatgagg ctctgcacaa ccactacacg cagaagagcc tctccctgtc tccgggttcc 1500

ggaggtggcg gttcgggagg tggcgggtca ggaggtgggg gatccccttc agaagtgcag 1560

ctgctggagt ccggaggagg actggtgcag cctggcggaa gcctgaggct gagctgcgct 1620

gcctccggct tcacattcag cagctatgct atgagctggg tgaggcaagc ccctggaaag 1680

tgcctggagt gggtgtccgc tatctccggc agcggcggaa gcacctacta cgctgactcc 1740

gtcaagggca ggttcaccat cagccgggac aacagcaaga acaccctgta cctgcagatg 1800

aatagcctca gggctgaaga caccgctgtg tactactgcg ccaggtacta tggcggctac 1860

tactccgcct ggatggacta ctggggacag ggcacactgg tgaccgtgtc cagcggcgga 1920

ggcggctccg gaggcggtgg ctccggagga ggcggaagcg gaggaggagg ctccgatatt 1980

cagatgacac agtcccctag ctccctgtcc gccagcgtgg gagatcgggt gaccatcacc 2040

tgcagggcca gccagtccat ctccagcttc ttaaactggt accagcagaa gcctggaaag 2100

gctcccaagc tgctgatcta cgccgcttcc agcctccaga gcggcgtgcc tagcaggttc 2160

tccggctccg gaagcggaac agacttcacc ctgaccatca gctccctgca gcccgaggac 2220

tccgctacct actactgcca gcagacctat ggctacctgc acaccttcgg ctgcggcaca 2280

aagctggaga tcaagcgc 2298

<210> 174

<211> 766

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - polypeptide construct to 5T4 x 4-1BB

<400> 174

Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro

1 5 10 15

Asp Thr Thr Gly Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val

20 25 30

Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr

35 40 45

Phe Ser His Gly Ser Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly

50 55 60

Leu Glu Trp Val Ser Ser Ile Ser Ser Gly Ser Gly Ser Thr Tyr Tyr

65 70 75 80

Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser His Asp Asn Ser Lys

85 90 95

Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala

100 105 110

Val Tyr Tyr Cys Ala Arg Ser Ser Tyr Tyr Gly Ser Tyr Tyr Ser Ile

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

145 150 155 160

Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val

165 170 175

Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser

180 185 190

Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu

195 200 205

Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser

210 215 220

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln

225 230 235 240

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Asp Asn Leu

245 250 255

Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Glu Pro Lys Ser

260 265 270

Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala

275 280 285

Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

290 295 300

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

305 310 315 320

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

325 330 335

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

340 345 350

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

355 360 365

Gly Lys Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala Leu Pro Ala Pro

370 375 380

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

385 390 395 400

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

405 410 415

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

420 425 430

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

435 440 445

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

450 455 460

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

465 470 475 480

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

485 490 495

Ser Pro Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

500 505 510

Gly Gly Ser Pro Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu

515 520 525

Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe

530 535 540

Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys

545 550 555 560

Cys Leu Glu Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr

565 570 575

Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

580 585 590

Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

595 600 605

Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Gly Gly Tyr Tyr Ser Ala Trp

610 615 620

Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly

625 630 635 640

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

645 650 655

Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser

660 665 670

Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser

675 680 685

Ser Phe Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu

690 695 700

Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe

705 710 715 720

Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu

725 730 735

Gln Pro Glu Asp Ser Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr Gly Tyr

740 745 750

Leu His Thr Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys Arg

755 760 765

<210> 175

<211> 2298

<212> DNA

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - polynucleotide construct for 5T4 x 4-1BB

<400> 175

atggaagcac cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccggt 60

gaggtgcagc tgttggagag cgggggaggc ttggtacagc ctggggggtc cctgcgcctc 120

tcctgtgcag ccagcggatt caccttttct cacggttcta tgtactgggt ccgccaggct 180

ccagggaagg ggctggagtg ggtctcatct atttcttctg gttctggttc tacatactat 240

gcagactccg tgaagggccg gttcaccatc tcccatgaca attccaagaa cacgctgtat 300

ctgcaaatga acagcctgcg tgccgaggac acggctgtat attattgtgc gcgctcttct 360

tactacggtt cttactactc tattgactat tggggccagg gaaccctggt caccgtctcc 420

tcaggtggag gtggctccgg gggtggaggt tccggaggag gcggatcagg tggaggcgga 480

agcgacatcc agatgaccca gtctccatcc tccctgagcg catctgtagg agaccgcgtc 540

accatcactt gccgggcaag tcagagcatt agcagctatt taaattggta tcagcagaaa 600

ccagggaaag cccctaagct cctgatctat gctgcatcca gtttgcaaag tggggtccca 660

tcacgtttca gtggcagtgg aagcgggaca gatttcactc tcaccatcag cagtctacaa 720

cctgaagatt ttgcaactta ttactgtcaa cagtactacg acaacctgcc cacttttggc 780

caggggacca agctggagat caaagagccc aaatcttctg acaaaactca cacatgccca 840

ccgtgcccag cacctgaagc cgcgggtgca ccgtcagtct tcctcttccc cccaaaaccc 900

aaggacaccc tcatgatctc ccggacccct gaggtcacat gcgtggtggt ggacgtgagc 960

cacgaagacc ctgaggtcaa gttcaactgg tacgtggacg gcgtggaggt gcataatgcc 1020

aagacaaagc cgcgggagga gcagtacaac agcacgtacc gtgtggtcag cgtcctcacc 1080

gtcctgcacc aggactggct gaatggcaag gaatacaagt gcgcggtctc caacaaagcc 1140

ctcccagccc ccatcgagaa aaccatctcc aaagccaaag ggcagccccg agaaccacag 1200

gtgtacaccc tgcccccatc ccgggatgag ctgaccaaga accaggtcag cctgacctgc 1260

ctggtcaaag gcttctatcc aagcgacatc gccgtggagt ggggagagcaa tgggcagccg 1320

gagaacaact acaagaccac gcctcccgtg ctggactccg acggctcctt cttcctctac 1380

agcaagctca ccgtggacaa gagcaggtgg cagcagggga acgtcttctc atgctccgtg 1440

atgcatgagg ctctgcacaa ccactacacg cagaagagcc tctccctgtc tccgggttcc 1500

ggaggtggcg gttcgggagg tggcgggtca ggaggtgggg gatccccttc agaagtgcag 1560

ctgctggagt ccggaggagg actggtgcag cctggcggaa gcctgaggct gagctgcgct 1620

gcctccggct tcacattcag cagctatgct atgagctggg tgaggcaagc ccctggaaag 1680

ggcctggagt gggtgtccgc tatctccggc agcggcggaa gcacctacta cgctgactcc 1740

gtcaagggca ggttcaccat cagccgggac aacagcaaga acaccctgta cctgcagatg 1800

aatagcctca gggctgaaga caccgctgtg tactactgcg ccaggtacta tggcggctac 1860

tactccgcct ggatggacta ctggggacag ggcacactgg tgaccgtgtc cagcggcgga 1920

ggcggctccg gaggcggtgg ctccggagga ggcggaagcg gaggaggagg ctccgatatt 1980

cagatgacac agtcccctag ctccctgtcc gccagcgtgg gagatcgggt gaccatcacc 2040

tgcagggcca gccagtccat ctccagctat ttaaactggt accagcagaa gcctggaaag 2100

gctcccaagc tgctgatcta cgccgcttcc agcctccaga gcggcgtgcc tagcaggttc 2160

tccggctccg gaagcggaac agacttcacc ctgaccatca gctccctgca gcccgaggac 2220

tccgctacct actactgcca gcagacctat ggctacctgc acaccttcgg ccagggcaca 2280

aagctggaga tcaagcgc 2298

<210> 176

<211> 766

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Obtained in the laboratory - polypeptide construct to 5T4 x 4-1BB

<400> 176

Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro

1 5 10 15

Asp Thr Thr Gly Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val

20 25 30

Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr

35 40 45

Phe Ser His Gly Ser Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly

50 55 60

Leu Glu Trp Val Ser Ser Ile Ser Ser Gly Ser Gly Ser Thr Tyr Tyr

65 70 75 80

Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser His Asp Asn Ser Lys

85 90 95

Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala

100 105 110

Val Tyr Tyr Cys Ala Arg Ser Ser Tyr Tyr Gly Ser Tyr Tyr Ser Ile

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

145 150 155 160

Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val

165 170 175

Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser

180 185 190

Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu

195 200 205

Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser

210 215 220

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln

225 230 235 240

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Asp Asn Leu

245 250 255

Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Glu Pro Lys Ser

260 265 270

Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala

275 280 285

Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

290 295 300

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

305 310 315 320

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

325 330 335

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

340 345 350

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

355 360 365

Gly Lys Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala Leu Pro Ala Pro

370 375 380

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

385 390 395 400

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

405 410 415

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

420 425 430

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

435 440 445

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

450 455 460

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

465 470 475 480

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

485 490 495

Ser Pro Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

500 505 510

Gly Gly Ser Pro Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu

515 520 525

Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe

530 535 540

Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys

545 550 555 560

Gly Leu Glu Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr

565 570 575

Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

580 585 590

Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

595 600 605

Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Gly Gly Tyr Tyr Ser Ala Trp

610 615 620

Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly

625 630 635 640

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

645 650 655

Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser

660 665 670

Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser

675 680 685

Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu

690 695 700

Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe

705 710 715 720

Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu

725 730 735

Gln Pro Glu Asp Ser Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr Gly Tyr

740 745 750

Leu His Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg

755 760 765

<210> 177

<211> 10

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDRH1 sequence ACN017

<400> 177

Ala Lys Gly Ser Gly Ser Tyr Phe Asp Leu

1 5 10

<210> 178

<211> 9

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDRL3 ACN017

<400> 178

Gln Gln Tyr Ser Gly Tyr Pro Tyr Thr

15

<210> 179

<211> 117

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> VH ACN017

<400> 179

Glu 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 Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly 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 Lys Gly Ser Gly Ser Tyr Phe Asp Leu Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 180

<211> 107

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> VL ACN017

<400> 180

Asp Ile Gln Met Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Gly Tyr Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 181

<211> 741

<212> PRT

<213> ARTIFICIAL SEQUENCE

<220>

<223> Full size ACN017

<400> 181

Glu 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 Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly 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 Lys Gly Ser Gly Ser Tyr Phe Asp Leu Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser

130 135 140

Pro Ser Phe Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys

145 150 155 160

Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys

165 170 175

Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln

180 185 190

Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

195 200 205

Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr

210 215 220

Cys Gln Gln Tyr Ser Gly Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys

225 230 235 240

Leu Glu Ile Lys Ser Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr

245 250 255

Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe

260 265 270

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

275 280 285

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

290 295 300

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

305 310 315 320

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

325 330 335

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

340 345 350

Ala Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

355 360 365

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

370 375 380

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

385 390 395 400

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

405 410 415

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

420 425 430

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

435 440 445

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

450 455 460

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser Gly Gly

465 470 475 480

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Pro Ser Glu

485 490 495

Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser

500 505 510

Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala

515 520 525

Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser

530 535 540

Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys

545 550 555 560

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu

565 570 575

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

580 585 590

Lys Gly Ser Gly Ser Tyr Phe Asp Leu Trp Gly Gln Gly Thr Leu Val

595 600 605

Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

610 615 620

Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro

625 630 635 640

Ser Phe Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg

645 650 655

Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro

660 665 670

Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser

675 680 685

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

690 695 700

Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

705 710 715 720

Gln Gln Tyr Ser Gly Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu

725 730 735

Glu Ile Lys Arg Ser

740

<---

Claims (28)

1. A bispecific polypeptide for use in the treatment of a disorder associated with 5T4 expression, comprising a first single chain variable fragment (scFv) domain and a second single chain variable fragment (scFv) domain linked together by a binding domain linker, and an immunoglobulin Fc domain, wherein said Fc the immunoglobulin domain contains a hinge region and an immunoglobulin constant region, wherein said bispecific polypeptide is capable of forming a homodimer by association with a second identical bispecific polypeptide, wherein said first scFv domain specifically binds to human 5T4 and contains: (i) an immunoglobulin heavy chain variable region comprising heavy chain complementarity determining region (HCDR) 1 with the amino acid sequence of SEQ ID NO: 30, HCDR2 with the amino acid sequence of SEQ ID NO: 32 and HCDR3 with the amino acid sequence of SEQ ID NO: 34; And (ii) an immunoglobulin light chain variable region comprising light chain complementarity determining region (LCDR) 1 with the amino acid sequence SEQ ID NO: 42, LCDR2 with the amino acid sequence SEQ ID NO: 10 and LCDR3 with the amino acid sequence SEQ ID NO: 36; And wherein said second scFv domain specifically binds to human 4-1BB and contains: (i) an immunoglobulin heavy chain variable region comprising HCDR1 with the amino acid sequence of SEQ ID NO: 2, HCDR2 with the amino acid sequence of SEQ ID NO: 4 and HCDR3 with the amino acid sequence of SEQ ID NO: 6; And (ii) an immunoglobulin light chain variable region comprising LCDR1 having the amino acid sequence SEQ ID NO: 8, LCDR2 having the amino acid sequence SEQ ID NO: 10 and LCDR3 having the amino acid sequence SEQ ID NO: 12. 2. A bispecific polypeptide according to claim 1, characterized in that said bispecific polypeptide contains, in the direction from the amino terminus to the carboxy terminus, (i) a first scFv domain, (ii) a hinge region, (iii) an immunoglobulin constant region, (iv) a linker binding domains and (v) a second scFv domain. 3. The bispecific polypeptide of claim 1, wherein the first scFv domain contains a mutation in the framework region compared to the framework region of SEQ ID NO: 130 or SEQ ID NO: 170. 4. Bispecific polypeptide according to claim 3, where the specified mutation leads to the appearance of a stabilizing disulfide bond. 5. The bispecific polypeptide of claim 1, wherein said first scFv domain comprises an immunoglobulin heavy chain variable region of the sequence SEQ ID NO: 38 and an immunoglobulin light chain variable region of the sequence SEQ ID NO: 44. 6. The bispecific polypeptide of claim 1, wherein said first scFv domain comprises an immunoglobulin heavy chain variable region of the sequence SEQ ID NO: 46 and an immunoglobulin light chain variable region of the sequence SEQ ID NO: 48. 7. The bispecific polypeptide of claim 1, wherein said second scFv domain comprises an immunoglobulin heavy chain variable region of the sequence SEQ ID NO: 14 and an immunoglobulin light chain variable region of the sequence SEQ ID NO: 16. 8. The bispecific polypeptide according to claim 1, wherein said first scFv domain comprises an immunoglobulin heavy chain variable region with the sequence SEQ ID NO: 46 and an immunoglobulin light chain variable region with the sequence SEQ ID NO: 48; and wherein said second scFv domain comprises an immunoglobulin heavy chain variable region of the sequence SEQ ID NO: 14 and an immunoglobulin light chain variable region of the sequence SEQ ID NO: 16. 9. The bispecific polypeptide of claim 1, wherein the amino acid sequence of said first scFv domain comprises SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 42, SEQ ID NO: 10 and SEQ ID NO : 36 and has at least 97% identity with SEQ ID NO: 120; and the amino acid sequence of said second scFv domain comprises SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12 and is at least 97% identity with SEQ ID NO: 110. 10. The bispecific polypeptide of claim 9, wherein said polypeptide comprises a first scFv domain comprising SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 42, SEQ ID NO: 10 and SEQ ID NO: 36, and a second scFv domain comprising SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12, and contains the amino acid sequence , which is at least 95% identical to the sequence of SEQ ID NO: 172; or contains an amino acid sequence that is identical to the sequence of SEQ ID NO: 172. 11. The bispecific polypeptide of claim 1, wherein the amino acid sequence of said first scFv domain comprises SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 42, SEQ ID NO: 10 and SEQ ID NO : 36 and has at least 97% identity with SEQ ID NO: 122; and the amino acid sequence of said second scFv domain comprises SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12 and is at least 97% identity with SEQ ID NO: 110. 12. The bispecific polypeptide of claim 11, wherein said polypeptide comprises a first scFv domain comprising SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 42, SEQ ID NO: 10, and SEQ ID NO: 36 and a second scFv domain comprising SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12, and contains the amino acid sequence , which is at least 95% identical to the sequence of SEQ ID NO: 174; or contains the amino acid sequence of SEQ ID NO: 174. 13. The bispecific polypeptide of claim 1, wherein binding of the bispecific polypeptide to an effector cell results in increased activation of the effector cell, increased proliferation of the effector cell, or wherein binding of the bispecific polypeptide to the effector cell and the 5T4-expressing cell enhances the effector cell-dependent lysis of said 5T4- expressing cell. 14. The bispecific polypeptide according to claim 1, wherein said polypeptide demonstrates statistically significantly enhanced activation of effector cells compared to the second bispecific polypeptide, wherein said second bispecific polypeptide is an IgG-scFv structure containing an antibody to 4-1BB containing a heavy chain variable region , containing SEQ ID NO: 28, and a light chain variable region, containing SEQ ID NO: 16, and scFv to 5T4, containing a heavy chain variable region, containing SEQ ID NO: 46, and a light chain variable region, containing SEQ ID NO: 66. 15. A bispecific polypeptide according to claim 1, wherein said polypeptide induces a statistically significantly increased proliferation of effector cells compared to a second bispecific polypeptide, wherein said second bispecific polypeptide is an IgG-scFv structure containing an antibody to 4-1BB containing a heavy chain variable region , containing SEQ ID NO: 28, and a light chain variable region, containing SEQ ID NO: 16, and scFv to 5T4, containing a heavy chain variable region, containing SEQ ID NO: 46, and a light chain variable region, containing SEQ ID NO: 66. 16. A method of treating cancer in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of the bispecific polypeptide of claim 1, wherein said cancer is characterized by expression of 5T4. 17. The method of claim 16, wherein said cancer is breast cancer, pancreatic cancer, ovarian cancer, non-small cell lung cancer, mesothelioma, chronic lymphocytic leukemia (CLL), mantle cell leukemia (MCL), acute lymphoblastic leukemia (ALL), squamous cell carcinoma, melanoma, adrenal cancer, bladder cancer, cervical cancer, kidney cancer, stomach cancer, prostate cancer, thyroid cancer, liver cancer, uterine cancer, neurofibroma, sarcoma, carcinoma or head and neck cancer. 18. The method of claim 16, wherein said bispecific polypeptide contains, in the amino-terminal to carboxy-terminal direction: (i) a first scFv domain, (ii) a hinge region, (iii) an immunoglobulin constant region, (iv) a linker binding domains, and (v) second scFv domain. 19. The method of claim 16, wherein said second scFv domain comprises an immunoglobulin heavy chain variable region of the sequence SEQ ID NO: 14 and an immunoglobulin light chain variable region of the sequence SEQ ID NO: 16, and wherein said first scFv domain contains: (i) an immunoglobulin heavy chain variable region having the sequence SEQ ID NO: 38, and an immunoglobulin light chain variable region having the sequence SEQ ID NO: 44; or (ii) an immunoglobulin heavy chain variable region of the sequence SEQ ID NO: 46, and an immunoglobulin light chain variable region of the sequence SEQ ID NO: 48. 20. The method of claim 16, wherein the amino acid sequence of said second scFv domain of said bispecific polypeptide comprises SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12 and has at least 97% identity with SEQ ID NO: 110; and wherein the amino acid sequence of said first scFv domain of said bispecific polypeptide comprises SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 42, SEQ ID NO: 10 and SEQ ID NO: 36 and has at least 97% identity with SEQ ID NO: 120 or SEQ ID NO: 122.