EA043303B1 - ANTIBODIES TO COLOGTING FACTOR XI - Google Patents

Description

Cross reference to related applications

This application claims priority to US Provisional Application No. 62/349888, filed June 14, 2016, which is incorporated herein by reference in its entirety.

Background of the Invention (1) Scope of the invention

The present invention provides antibodies that bind to the apple 3 domain of human coagulation factor XI (FXI) and inhibit the activation of FXI by coagulation factor XIIa, as well as the action of FXIa on factor IX (FIX).

(2) Description of the prior art

Thromboembolic disorders, which include both venous and arterial thrombosis, remain a leading cause of morbidity and mortality in the Western world, despite the availability of a large class of anticoagulants such as vitamin K antagonists (VKAs), heparins, and direct thrombin inhibitors (Weitz et al., Chest 2008, 133: 234S-256S; Hawkins, Pharmacotherapy 2004, 24:62S65S). These drugs are effective in reducing the risk of thrombosis, but they are associated with many limitations. For example, VKAs (eg, warfarin) have been the mainstay of oral anticoagulation, although management of VKA therapy is complicated by the significant risk of bleeding, slow onset and offset of action, and multiple food-drug interactions (Hawkins, in citation; Ansell J et al. , Chest 2008, 133:160S-198S). Nonvitamin K antagonist oral anticoagulants (NOACs, including rivaroxaban, apixaban, edoxaban, and dabigatran) demonstrate at least noninferiority to warfarin, with fewer food-drug interactions, and do not require monitoring. However, DOACs still increase the risk of bleeding, as evidenced by the nearly 15% annual incidence of clinically significant or nonsignificant bleeding in the registration studies of stroke prevention in atrial fibrillation (Connolly et al., N Engl J Med 2009, 361:1139-1151; Patel et al., N Engl J Med 2011, 365:883-891; Granger et al., N Engl J Med 2011, 365:981-992; Giugliano et al., N Engl J Med 2013, 369:2093-2104 ). This is largely due to the fact that the target proteins of NOAC (clotting factor Xa (FXa) and thrombin) are required for normal clotting (hemostasis). Thus, new therapies with better safety profiles are in demand for the prevention and treatment of thrombotic diseases or disorders.

In the classic cascade model (waterfall model) of blood coagulation (Fig. 1A), coagulation is initiated either by the extrinsic pathway (activated tissue factor (TF)) or the intrinsic pathway (contact activation); both lead to a common pathway that culminates in the formation of thrombin and fibrin (Furie & Furie, Cell 1988, 53:505-518; Gailani & Renne, J Thromb Haemost 2007, 5:11061112). The extrinsic cascade is triggered when TF, which is present in the subendothelium and atherosclerotic plaques, is exposed to flowing blood and forms a complex with coagulation factor VIIa (FVIIa). The TF-FVIIa complex (extrinsic tenase complex) then initiates the general pathway, i.e. activation of FX to form Fxa, which in turn converts prothrombin to thrombin. The TF-FVIIa complex can also activate coagulation factor IX (FIX) to form FIXa. FIXa in complex with coagulation factor VIII (FVIIIa) (intrinsic tenase complex) can also cleave FX substrate. The intrinsic cascade is initiated when FXIIa is formed by contact activation with negatively charged surfaces (eg, collagen and glycosaminoglycans) and thrombin generation propagates through the sequential activation of FXI, FIX, FX, and prothrombin. Thrombin, as the final protease in the coagulation cascade, may further contribute to the formation of FXIa through direct feedback activation of FXI. Platelets, another important coagulation component in whole blood, can be activated by thrombin and can then also support the formation of FXIa. FXI-dependent propagation of thrombin generation may indirectly regulate fibrinolysis through activation of thrombin-activated fibrinolysis inhibitor (ATIF). FXI thus interacts with several components in the hemostatic system and plays a major role in blood coagulation and thrombosis (Gailani & Renne, op.cit; Emsley et al., Blood 2010, 115:2569-2577).

Factor XI (FXI) is a dimer consisting of identical 80 kDa subunits, and each subunit, starting at the N terminus, consists of four apple domains (A1, A2, A3, and A4) and a catalytic domain (See FIG. .1B). FXI is a zymogen that circulates in complex with high molecular weight kinogen (MC). BK binds to the A2 domain of FXI and is a physiological cofactor of FXIIa to activate FXI to FXIa. The remaining apple domains in FXI also mediate important physiological functions. For example, the FIX-binding external site is located in A3, while the FXIIa binding site is located in A4. Residues that are important for FXI dimerization are also located in A4 (Emsley et al., op. cit.).

In recent years, several lines of work have demonstrated that FXI plays a major role in the pathological process of thrombus formation with a relatively minor contribution to hemostasis and thus represents a promising target in thrombosis. Key data supporting this view are summarized below: (1) a phase II antisense oligonucleotide (ASO) study

- 1 043303 for FXI from lonis Pharmaceutics Inc. (Buller et al., N Engl J Med 2015, 372:232-240), ASO for FXI caused a significant reduction in venous thromboembolism (VTE) with a trend toward less bleeding compared with enoxaparin in patients who underwent total knee arthroplasty; (2) Human genetics and epidemiology studies (Duga et al., Semin Thromb Hemost 2013; Chen et al., Drug Discov Today 2014; Key, Hematology Am Soc Hematol Educ 2014 Program 2014:66-70) indicate that severe FXI deficiency (hemophilia C) is associated with a reduced risk of ischemic stroke and deep vein thrombosis; in contrast, elevated FXI levels are associated with a higher risk of VTE and ischemic stroke; and (3) a number of lines of preclinical research have demonstrated that inhibition or loss of FXI(a) function mediates significant protection against thrombus without compromising hemostasis (Chen et al. op. cit.). Of note, monoclonal antibodies 14E11 and 1A6 produce significant thrombus reduction in the baboon arteriovenous shunt thrombosis model (US Pat. No. 8,388,959; US Pat. No. 8,236,316; Tucker et al., Blood 2009, 113:936-944; Cheng et al., Blood 2010, 116:3981-3989). In addition, 14E11 (as it cross-reacts with murine FXI) was protective in a mouse model of acute ischemic stroke (Leung et al., Transl Stroke Res 2012, 3:381-389). Additional mAbs targeting FXI have also been described in preclinical models to validate FXI as an antithrombotic target with minimal bleeding risk (van Montfoort et al., Thromb Haemost 2013, 110; Takahashi et al., Thromb Res 2010, 125:464- 470; van Montfoort, Ph.D. Thesis, University of Amsterdam, Amsterdam, Netherlands, 14 November 2014). Inhibition of FXI thus represents a promising strategy for novel antithrombotic therapy with an improved benefit-risk profile compared with current standard of care anticoagulants.

There is currently a large unmet medical need for antithrombotic therapy for patients with severe or end-stage renal disease (ESRD). Approximately 650,000 patients in the United States have severe or end-stage renal failure, and these patients suffer from an extremely high incidence of thrombotic and thromboembolic complications (myocardial infarction, stroke/transient ischemic attack, peripheral arterial disease (PAD), vascular access disorder). Patients with ESRD are also more likely to have bleeding events than the general population. Because patients with ESRD are not routinely prescribed any type of anticoagulation (due to the risk of bleeding and the lack of data for non-vitamin K antagonist oral coagulants (NVKAs) in ESRD), there is a need for antithrombotic therapy that has an acceptable benefit profile. risk in these patients.

Brief summary of the invention

The present invention relates to human antibodies capable of selectively binding to coagulation factor XI (anti-FXI antibodies) and inhibiting blood coagulation and associated thrombosis, preferably without affecting hemostasis. The compositions include anti-coagulation factor XI antibodies capable of binding to a specific epitope of the apple 3 (A3) domain of coagulation factor XI. These antibodies exhibit neutralizing activity by inhibiting the conversion of the zymogen form of FXI to its activated form, FXIa, by FXIIa, and by inhibiting FXIa-mediated activation of FIX. Antibodies are suitable for inhibition of FXI, which may have a clinically significant antithrombotic effect with a reduced risk of bleeding, and thus may have an expanded therapeutic index compared to inhibition of downstream coagulation factors such as FXa and thrombin. Thus, these antibodies provide a therapeutic approach for the prevention of thromboembolic complications, such as stroke prevention in atrial fibrillation (SPAF).

One unserved cohort at risk for vascular thrombosis that may benefit from FXI inhibition is the population with severe and end-stage renal disease (ESRD) in which non-vitamin K antagonist oral anticoagulants (NVKAs) are not typically used. due to bleeding-related reasons, which has led to a lack of clinical trial experience. The antibodies herein provide a novel anticoagulant therapy for the prevention of thrombotic complications in patients with ESRD. The antibodies herein may provide clinically significant antithrombotic efficacy with an acceptable bleeding risk in patients with ESRD.

In addition to ESRD and SPAF, FXI inhibition may also be indicated in additional patient segments at high risk of thrombosis. These include:

1) venous thromboembolism (VTE) prevention during orthopedic surgery and/or secondary prevention of VTE;

2) reduction of revascularization and/or reduction of major adverse limb complications (MALE) in peripheral arterial atherosclerosis (PAA);

3) adjuvant therapy for acute coronary syndrome (ACS).

The present invention relates to an antibody or antigen binding fragment thereof comprising at least six complementarity determining regions (CDRs) from an anti-FXI antibody of the aFXI-18623p family, the aFXI-18611p family or the aFXI-18611 family, or at least six

- 2 043303 complementarity determining regions (CDRs) from an anti-FXI antibody of the aFXI-18623p family, the aFXI-18611p family, or the aFXI-18611 family, where one or more of the six CDRs have one, two, or three amino acid substitutions, insertions, deletions, or combinations thereof, wherein the aFXI-18623 family antibody contains a heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 28 or 29, and a light chain variable region with the amino acid sequence shown in SEQ ID NO: 30; an antibody of the aFXI-18611p family contains a heavy chain variable region with the amino acid sequence shown in SEQ ID NO:21 or 22, and a light chain variable region with the amino acid sequence shown in SEQ ID NO:25; and an antibody of the aFXI-18611 family comprises a heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 23 or 24, and a light chain variable region with the amino acid sequence shown in SEQ ID NO: 25. In further embodiments, the antibody or antigen-binding fragment binds to the apple 3 domain of coagulation factor XI (FXI) and inhibits activation of FXI and/or factor XIa-mediated activation of factor IX.

In additional aspects or embodiments of the invention, the six CDRs include or comprise the heavy chain CDR1, CDR2, and CDR3 of an anti-FXI antibody from the aFXI-18623p family, the aFXI18611p family, or the aFXI-18611 family, and the light chain CDR1, CDR2, and CDR3 of an antibody from the aFXI18623p family , aFXI-18611p family or aFXI-18611 family, wherein the aFXI-118623 family antibody contains a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 28 or 29, and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 30; an antibody of the aFXI-18611p family contains a heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 21 or 22, and a light chain variable region with the amino acid sequence shown in SEQ ID NO: 25; and an antibody of the aFXI-18611 family comprises a heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 23 or 24, and a light chain variable region with the amino acid sequence shown in SEQ ID NO: 25. In further embodiments, the antibody or antigen-binding fragment binds to the apple 3 domain of coagulation factor XI (FXI) and inhibits activation of FXI and/or factor XIa-mediated activation of factor IX.

In additional aspects or embodiments of the invention, the antibody or antigen binding fragment comprises a heavy chain variable region with an amino acid sequence selected from the group of amino acid sequences consisting of SEQ ID NOs: 21, 22, 23, and 24; and a light chain variable region with the amino acid sequence shown in SEQ ID NO: 25; wherein the heavy chain variable region framework may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, insertions, deletions, or combinations thereof, and the light chain variable region framework may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, insertions, deletions, or combinations thereof.

In additional aspects or embodiments of the invention, the antibody or antigen binding fragment comprises a heavy chain variable region with an amino acid sequence selected from the group of amino acid sequences consisting of SEQ ID NOs: 21, 22, 23, and 24; and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 25.

In additional aspects or embodiments of the invention, the antibody or antigen binding fragment comprises a heavy chain variable region with an amino acid sequence selected from the group of amino acid sequences consisting of SEQ ID NOs: 28 and 29; and a light chain variable region with the amino acid sequence shown in SEQ ID NO: 30; wherein the heavy chain variable region framework may contain 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, insertions, deletions, or combinations thereof, and the light chain variable region framework may contain 1, 2, 3 , 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, insertions, deletions, or combinations thereof.

In additional aspects or embodiments of the invention, the antibody or antigen binding fragment comprises a heavy chain variable region with an amino acid sequence selected from the group of amino acid sequences consisting of SEQ ID NOs: 28 and 29; and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 30.

In additional aspects or embodiments of the invention, the antibody comprises a heavy chain constant domain of a human IgG1, IgG2, IgG3, or IgG4 isotype. In additional aspects, the constant domain may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, insertions, deletions, or combinations thereof. In particular aspects, the constant domain may or may not contain a C-terminal lysine.

In additional aspects or embodiments of the invention, the antibody comprises a heavy chain constant domain of a human IgG1 or IgG4 isotype. In a further aspect, the heavy chain constant domain is of the IgG4 isotype and further comprises replacing the serine residue at position 228 (EU numbering) with a proline, corresponding to position 108 of SEQ ID NO: 16 or 17 (serine at position 108).

- 3 043303

In additional aspects or embodiments of the invention, the antibody comprises a heavy chain constant domain comprising the amino acid sequence shown in SEQ ID

NO: 16, 17, 18, or 19.

In additional aspects or embodiments of the invention, the antibody comprises a human kappa or lambda light chain constant domain.

In further aspects or embodiments of the invention, the antibody comprises a light chain constant domain comprising the amino acid sequence shown in SEQ ID NO: 20.

In additional aspects or embodiments of the invention, the antibody or antigen binding fragment comprises a heavy chain with an amino acid sequence selected from the group of amino acid sequences consisting of SEQ ID NO: 33, 35, 37, 39, 45, 47, 49, 51, 57, 59 , 61, 63, 69, 71, 73 and 75; and a light chain with the amino acid sequence shown in SEQ ID NO: 26.

In additional aspects or embodiments of the invention, the antibody or antigen binding fragment comprises a heavy chain with an amino acid sequence selected from the group of amino acid sequences consisting of SEQ ID NO: 41, 43, 53, 55, 65, 67, 77 and 79; and a light chain with the amino acid sequence shown in SEQ ID NO: 31.

The present invention further provides an antibody or antigen binding fragment comprising: (a) a heavy chain variable domain having the amino acid sequence shown in SEQ ID NO: 28 and a light chain variable domain having the amino acid sequence shown in SEQ ID NO: 30; (b) a heavy chain variable domain having the amino acid sequence shown in SEQ ID NO: 29 and a light chain variable domain having the amino acid sequence shown in SEQ ID NO: 30; (b) a heavy chain variable domain having the amino acid sequence shown in SEQ ID NO: 21 and a light chain variable domain having the amino acid sequence shown in SEQ ID NO: 25; (c) a heavy chain variable domain having the amino acid sequence shown in SEQ ID NO: 22 and a light chain variable domain having the amino acid sequence shown in SEQ ID NO: 25; (d) a heavy chain variable domain having the amino acid sequence shown in SEQ ID NO: 23 and a light chain variable domain having the amino acid sequence shown in SEQ ID NO: 25, or (e) a heavy chain variable domain having the amino acid sequence shown in SEQ ID NO: 24, and a light chain variable domain having the amino acid sequence shown in SEQ ID NO: 25.

In additional embodiments, the antibody or antigen-binding fragment binds to the apple 3 domain of coagulation factor XI (FXI) and inhibits activation of FXI and/or factor XIa-mediated activation of factor IX.

In specific embodiments, the heavy and light chain variable regions may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, insertions, deletions, or combinations thereof.

In specific embodiments, the heavy and light chain constant domains may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, insertions, deletions, or combinations thereof. In certain aspects, the constant domain may or may not contain a C-terminal lysine.

In specific embodiments, the heavy and light chain variable regions may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, insertions, deletions, or combinations thereof, and the heavy and light chain constant domains may contain 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, insertions, deletions, or combinations thereof. In certain aspects, the constant domain may or may not contain a C-terminal lysine.

In additional aspects or embodiments of the invention, the antibody further comprises a heavy chain constant domain comprising the amino acid sequence shown in SEQ ID NO: 16, 17, 18, or 19 or a variant thereof comprising 1, 2, 3, 4, 5, 6 , 7, 8, 9 or 10 amino acid substitutions, insertions, deletions, or combinations thereof.

In additional aspects or embodiments of the invention, the antibody further comprises a light chain constant domain comprising the amino acid sequence shown in SEQ ID NO:20 or a variant thereof comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, insertions, deletions, or combinations thereof.

In a further aspect or embodiment of the invention, the antibody or antigen binding fragment comprises:

(a) a heavy chain variable domain having the amino acid sequence shown in SEQ ID NO: 28 and a light chain variable domain having the amino acid sequence shown in SEQ ID NO: 30; (b) a heavy chain variable domain having the amino acid sequence shown in SEQ ID NO: 29 and a light chain variable domain having the amino acid sequence shown in SEQ ID NO: 30; (c) a heavy chain variable domain having the amino acid sequence shown in SEQ ID NO: 21 and a light chain variable domain having the amino acid sequence shown in SEQ ID NO: 25; (d) a heavy chain variable domain with the amino acid sequence shown in SEQ ID NO: 22, and a light chain variable domain with the amino acid sequence

- 4 043303 sequence shown in SEQ ID NO: 25; (e) a heavy chain variable domain having the amino acid sequence shown in SEQ ID NO: 23 and a light chain variable domain having the amino acid sequence shown in SEQ ID NO: 25; (f) a heavy chain variable domain having the amino acid sequence shown in SEQ ID NO: 24 and a light chain variable domain having the amino acid sequence shown in SEQ ID NO: 25; (g) option (a), (b), (c), (d), (e), or (f), wherein the heavy chain variable region framework contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, insertions, deletions or combinations thereof; or (h) option (a), (b), (c), (d), (e), (f) or (g), wherein the light chain variable region framework contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, insertions, deletions, or combinations thereof.

The present invention further provides an antibody comprising: (a) a heavy chain (HC) with a constant domain and a variable domain, wherein the variable domain comprises a heavy chain complementarity determining region (HC-CDR) 1 with the amino acid sequence shown in SEQ ID NO: 1 , HC-CDR 2 with the amino acid sequence shown in SEQ ID NO: 2, and HC-CDR 3 with the amino acid sequence shown in SEQ ID NO: 3; (b) a heavy chain (HC) with a constant domain and a variable domain, where the variable domain contains a heavy chain complementarity determining region (HC-CDR) 1 with the amino acid sequence shown in SEQ ID NO: 1, HC-CDR 2 with the amino acid sequence, shown in SEQ ID NO: 2, and HC-CDR 3 with the amino acid sequence shown in SEQ ID NO: 4; or (c) a heavy chain (HC) with a constant domain and a variable domain, wherein the variable domain comprises a heavy chain complementarity determining region (HC-CDR) 1 with the amino acid sequence shown in SEQ ID NO: 8, HC-CDR 2 with the amino acid sequence shown in SEQ ID NO: 9, and HC-CDR 3 with the amino acid sequence shown in SEQ ID NO: 10. In additional embodiments, the antibody or antigen binding fragment binds to the apple 3 domain of coagulation factor XI (FXI) and inhibits activation of FXI and /or factor XIa-mediated activation of factor IX.

In additional aspects or embodiments of the invention, the antibody comprises a heavy chain constant domain of a human IgG1, IgG2, IgG3, or IgG4 isotype. In additional aspects, the constant domain may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, insertions, deletions, or combinations thereof, compared to the amino acid sequence of the native heavy chain constant domain for the IgG1 isotype. Human IgG2, IgG3, or IgG4. In certain aspects, the constant domain may or may not contain a C-terminal lysine.

In additional aspects or embodiments of the invention, the antibody comprises a human IgG1 or IgG4 isotype heavy chain constant domain. In a further aspect, the heavy chain constant domain is of the IgG4 isotype and further comprises replacing the serine residue at position 228 (EU numbering) with a proline, corresponding to position 108 of SEQ ID NO:16 or 17 (serine at position 108).

In additional aspects or embodiments of the invention, the antibody comprises an IgG4 heavy chain constant domain comprising the amino acid sequence shown in SEQ ID NO: 16 or 17. In additional aspects, the constant domain may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, insertions, deletions, or combinations thereof.

In additional aspects or embodiments of the invention, the antibody comprises an IgG1 heavy chain constant domain comprising the amino acid sequence shown in SEQ ID NO: 18 or 19. In additional aspects, the constant domain may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, insertions, deletions, or combinations thereof.

The present invention further provides an antibody or antigen binding fragment comprising:

(a) a light chain (LC) with a constant domain and a variable domain, wherein the variable domain contains a light chain complementarity determining region (LC-CDR) 1 with the amino acid sequence shown in SEQ ID NO: 5, LC-CDR 2 with the amino acid sequence, shown in SEQ ID NO: 6, and LC-CDR 3 with the amino acid sequence shown in SEQ ID NO: 7; or (b) a light chain (LC) with a constant domain and a variable domain, wherein the variable domain comprises a light chain complementarity determining region (LC-CDR) 1 with the amino acid sequence shown in SEQ ID NO: 11, LC-CDR 2 with the amino acid sequence shown in SEQ ID NO: 12, and LC-CDR 3 with the amino acid sequence shown in SEQ ID NO: 13. In additional embodiments, the antibody or antigen binding fragment binds to the apple 3 domain of coagulation factor XI (FXI) and inhibits activation of FXI and /or factor XIa-mediated activation of factor IX.

In additional aspects or embodiments of the invention, the light chain (LC) comprises human kappa light chain or human lambda light chain, or a variant thereof containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, insertions , amino acid deletions, or combinations thereof, where the antibody or antigen-binding fragment binds to the apple 3 domain of coagulation factor XI (FXI) and inhibits activation

- 5 043303

FXI and/or factor XIa-mediated activation of factor IX. In further aspects or embodiments of the invention, the antibody comprises a light chain constant domain comprising the amino acid sequence shown in SEQ ID NO: 20.

In additional aspects or embodiments of the invention, the antibody comprises an IgG4 heavy chain constant domain comprising the amino acid sequence shown in SEQ ID NO: 16 or 17, or a variant thereof comprising 1, 2, 3, 4, 5, 6, 7, 8 , 9 or 10 amino acid substitutions, insertions, deletions, or combinations thereof, wherein the antibody or antigen binding fragment binds to the apple 3 domain of coagulation factor XI (FXI) and inhibits activation of FXI and/or factor XIa-mediated activation of factor IX.

In additional aspects or embodiments of the invention, the antibody comprises an IgG1 heavy chain constant domain comprising the amino acid sequence shown in SEQ ID NO: 18 or 19, or a variant thereof comprising 1, 2, 3, 4, 5, 6, 7, 8 , 9 or 10 amino acid substitutions, insertions, deletions, or combinations thereof, wherein the antibody or antigen binding fragment binds to the apple 3 domain of coagulation factor XI (FXI) and inhibits activation of FXI and/or factor XIa-mediated activation of factor IX.

The present invention further provides an antibody or antigen binding fragment comprising:

(a) a heavy chain (HC) with a constant domain and a variable domain, where the variable domain contains a heavy chain complementarity determining region (HC-CDR) 1 with the amino acid sequence shown in SEQ ID NO: 1, HC-CDR 2 with the amino acid sequence, shown in SEQ ID NO: 2, and HC-CDR 3 with the amino acid sequence shown in SEQ ID NO: 3; and (b) a light chain (LC) with a constant domain and a variable domain, wherein the variable domain comprises a light chain complementarity determining region (LC-CDR) 1 with the amino acid sequence shown in SEQ ID NO: 5, LC-CDR 2 with the amino acid sequence shown in SEQ ID NO: 6, and LC-CDR 3 with the amino acid sequence shown in SEQ ID NO: 7. In additional embodiments, the antibody or antigen binding fragment binds to the apple 3 domain of coagulation factor XI (FXI) and inhibits activation of FXI and /or factor XIa-mediated activation of factor IX.

In additional aspects or embodiments of the invention, the light chain (LC) comprises human kappa light chain or human lambda light chain, or a variant thereof containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, insertions, deletions of amino acids, or combinations thereof, wherein the antibody or antigen-binding fragment binds to the apple 3 domain of coagulation factor XI (FXI) and inhibits activation of FXI and/or factor XIa-mediated activation of factor IX. In further aspects or embodiments of the invention, the antibody comprises a light chain constant domain comprising the amino acid sequence shown in SEQ ID NO: 20.

In additional aspects or embodiments of the invention, the antibody contains a heavy chain constant domain of an IgG1, IgG2, IgG3, or IgG4 isotype, or a variant thereof containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, insertions, deletions of amino acids or combinations thereof compared to the amino acid sequence of the native IgG1, IgG2, IgG3, or IgG4 isotype, wherein the antibody or antigen-binding fragment binds to the apple 3 domain of coagulation factor XI (FXI) and inhibits activation of FXI and/or factor XIa-mediated activation of factor IX . In additional aspects, the constant domain may or may not contain a C-terminal lysine.

In additional aspects or embodiments of the invention, the antibody comprises a human IgG1 or IgG4 isotype heavy chain constant domain or variant thereof comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, insertions, deletions, or combinations thereof, wherein the antibody or antigen-binding fragment binds to the apple 3 domain of coagulation factor XI (FXI) and inhibits activation of FXI and/or factor XIa-mediated activation of factor IX. In a further aspect, the heavy chain constant domain is of the IgG4 isotype and further includes the replacement of the serine residue at position 228 (EU numbering) with a proline, corresponding to position 108 of SEQ ID NO: 16 or 17 (serine at position 108).

In additional aspects or embodiments of the invention, the antibody comprises an IgG4 heavy chain constant domain comprising the amino acid sequence shown in SEQ ID NO:16 or 17, or a variant thereof comprising 1, 2, 3, 4, 5, 6, 7, 8 , 9 or 10 amino acid substitutions, insertions, deletions, or combinations thereof, wherein the antibody or antigen binding fragment binds to the apple 3 domain of coagulation factor XI (FXI) and inhibits activation of FXI and/or factor XIa-mediated activation of factor IX.

In additional aspects or embodiments of the invention, the antibody comprises an IgG1 heavy chain constant domain comprising the amino acid sequence shown in SEQ ID NO: 18 or 19, or a variant thereof comprising 1, 2, 3, 4, 5, 6, 7, 8 , 9 or 10 amino acid substitutions, insertions, deletions, or combinations thereof, wherein the antibody or antigen binding fragment binds to the apple 3 domain of coagulation factor XI (FXI) and inhibits FXI and/or factor XIa-mediated activation

- 6 043303 activation of factor IX.

The present invention further provides an antibody or antigen binding fragment comprising:

(a) a heavy chain (HC) with a constant domain and a variable domain, where the variable domain contains a heavy chain complementarity determining region (HC-CDR) 1 with the amino acid sequence shown in SEQ ID NO: 1, HC-CDR 2 with the amino acid sequence, shown in SEQ ID NO: 2, and HC-CDR 3 with the amino acid sequence shown in SEQ ID NO: 4; and (b) a light chain (LC) with a constant domain and a variable domain, wherein the variable domain comprises a light chain complementarity determining region (LC-CDR) 1 with the amino acid sequence shown in SEQ ID NO: 5, LC-CDR 2 with the amino acid sequence shown in SEQ ID NO: 6, and LC-CDR 3 with the amino acid sequence shown in SEQ ID NO: 7. In additional embodiments, the antibody or antigen binding fragment binds to the apple 3 domain of coagulation factor XI (FXI) and inhibits activation of FXI and /or factor XIa-mediated activation of factor IX.

In further aspects or embodiments of the invention, the light chain comprises a human kappa light chain or a human lambda light chain or a variant thereof comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, insertions, deletions or combinations thereof, wherein the antibody or antigen-binding fragment binds to the apple 3 domain of coagulation factor XI (FXI) and inhibits activation of FXI and/or factor XIa-mediated activation of factor IX. In further aspects or embodiments of the invention, the antibody comprises a light chain constant domain comprising the amino acid sequence shown in SEQ ID NO: 20.

In additional aspects or embodiments of the invention, the antibody comprises a heavy chain constant domain of an IgG1, IgG2, IgG3, or IgG4 isotype, or a variant thereof comprising 1, 2, 3, 4,

5, 6, 7, 8, 9, or 10 amino acid substitutions, insertions, deletions, or combinations thereof compared to the amino acid sequence of the native IgG1, IgG2, IgG3, or IgG4 isotype, where the antibody or antigen-binding fragment binds to the apple 3 domain of coagulation factor XI ( FXI) and inhibits FXI activation and/or factor XIa-mediated activation of factor IX. In additional aspects, the constant domain may or may not contain a C-terminal lysine.

In additional aspects or embodiments of the invention, the antibody comprises a human IgG1 or IgG4 isotype heavy chain constant domain or variant thereof comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, insertions, deletions, or combinations thereof, wherein the antibody or antigen-binding fragment binds to the apple 3 domain of coagulation factor XI (FXI) and inhibits activation of FXI and/or factor XIa-mediated activation of factor IX. In a further aspect, the heavy chain constant domain is of the IgG4 isotype and further includes the replacement of the serine residue at position 228 (EU numbering) with a proline, corresponding to position 108 of SEQ ID NO: 16 or 17 (serine at position 108).

In additional aspects or embodiments of the invention, the antibody comprises an IgG4 heavy chain constant domain comprising the amino acid sequence shown in SEQ ID NO:16 or 17, or a variant thereof comprising 1, 2, 3, 4, 5, 6, 7, 8 , 9 or 10 amino acid substitutions, insertions, deletions, or combinations thereof, wherein the antibody or antigen binding fragment binds to the apple 3 domain of coagulation factor XI (FXI) and inhibits activation of FXI and/or factor XIa-mediated activation of factor IX.

In additional aspects or embodiments of the invention, the antibody comprises an IgG1 heavy chain constant domain comprising the amino acid sequence shown in SEQ ID NO: 18 or 19, or a variant thereof comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, insertions, deletions, or combinations thereof, wherein the antibody or antigen binding fragment binds to the apple 3 domain of coagulation factor XI (FXI) and inhibits activation of FXI and/or factor XIa-mediated activation of factor IX.

The present invention further provides an antibody or antigen binding fragment comprising:

(a) a heavy chain (HC) with a constant domain and a variable domain, where the variable domain contains a heavy chain complementarity determining region (HC-CDR) 1 with the amino acid sequence shown in SEQ ID NO: 8, HC-CDR 2 with the amino acid sequence, shown in SEQ ID NO: 9, and HC-CDR 3 with the amino acid sequence shown in SEQ ID NO: 10; and (b) a light chain (LC) with a constant domain and a variable domain, wherein the variable domain comprises a light chain complementarity determining region (LC-CDR) 1 with the amino acid sequence shown in SEQ ID NO: 11, LC-CDR 2 with the amino acid sequence shown in SEQ ID NO: 12, and LC-CDR 3 with the amino acid sequence shown in SEQ ID NO: 13. In additional embodiments, the antibody or antigen binding fragment binds to the apple 3 domain of coagulation factor XI (FXI) and inhibits activation of FXI and /or indirectly-

- 7 043303 factor XIa activation of factor IX.

In further aspects or embodiments of the invention, the light chain comprises a human kappa light chain or a human lambda light chain or a variant thereof comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, insertions, deletions or combinations thereof, wherein the antibody or antigen-binding fragment binds to the apple 3 domain of coagulation factor XI (FXI) and inhibits activation of FXI and/or factor XIa-mediated activation of factor IX. In further aspects or embodiments of the invention, the antibody comprises a light chain constant domain comprising the amino acid sequence shown in SEQ ID NO: 20.

In additional aspects or embodiments of the invention, the antibody comprises an IgG1, IgG2, IgG3, or IgG4 isotype heavy chain constant domain or a variant thereof containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, insertions, deletions of amino acids or combinations thereof compared to the amino acid sequence of the native IgG1, IgG2, IgG3, or IgG4 isotype, wherein the antibody or antigen-binding fragment binds to the apple 3 domain of coagulation factor XI (FXI) and inhibits activation of FXI and/or factor XIa-mediated activation of factor IX . In additional aspects, the constant domain may or may not contain a C-terminal lysine.

In additional aspects or embodiments of the invention, the antibody comprises a human IgG1 or IgG4 isotype heavy chain constant domain or variant thereof comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, insertions, deletions, or combinations thereof, wherein the antibody or antigen-binding fragment binds to the apple 3 domain of coagulation factor XI (FXI) and inhibits activation of FXI and/or factor XIa-mediated activation of factor IX. In a further aspect, the heavy chain constant domain is of the IgG4 isotype and further comprises replacing the serine residue at position 228 (EU numbering) with a proline, corresponding to position 108 of SEQ ID NO: 16 or 17 (serine at position 108).

In additional aspects or embodiments of the invention, the antibody comprises an IgG4 heavy chain constant domain comprising the amino acid sequence shown in SEQ ID NO: 16 or 17, or a variant thereof comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, insertions, deletions, or combinations thereof, wherein the antibody or antigen binding fragment binds to the apple 3 domain of coagulation factor XI (FXI) and inhibits activation of FXI and/or factor XIa-mediated activation of factor IX.

In additional aspects or embodiments of the invention, the antibody comprises an IgG1 heavy chain constant domain comprising the amino acid sequence shown in SEQ ID NO: 18 or 19, or a variant thereof comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, insertions, deletions, or combinations thereof, wherein the antibody or antigen binding fragment binds to the apple 3 domain of coagulation factor XI (FXI) and inhibits activation of FXI and/or factor XIa-mediated activation of factor IX.

In further aspects or embodiments, the present invention provides an antibody comprising: (a) a heavy chain (HC) with a constant domain and a variable domain, wherein the variable domain comprises (i) a HC framework and a heavy chain complementarity determining region (HC-CDR) 1 with the amino acid sequence shown in SEQ ID NO: 8, HC-CDR 2 with the amino acid sequence shown in SEQ ID NO: 9, and HC-CDR 3 with the amino acid sequence shown in SEQ ID NO: 10; (ii) an HC framework and heavy chain complementarity determining region (HC-CDR) 1 with the amino acid sequence shown in SEQ ID NO: 1, HC-CDR 2 with the amino acid sequence shown in SEQ ID NO: 2, and HC-CDR 3 with the amino acid sequence shown in SEQ ID NO: 3; (iii) an HC framework and heavy chain complementarity determining region (HC-CDR) 1 with the amino acid sequence shown in SEQ ID NO: 1, HC-CDR 2 with the amino acid sequence shown in SEQ ID NO: 2, and HCCDR 3 with the amino acid the sequence shown in SEQ ID NO: 4; (iv) option (i), (ii), or (iii), where at least one of HC CDR 1, HC-CDR 2, or CDR 3 contains 1, 2 or 3 amino acid substitutions, insertions, deletions, or combinations thereof ; or (v) option (i), (ii), (iii), or (iv), where the NS framework contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, insertions, deletions amino acids or combinations thereof; (b) a light chain (LC) with a constant domain and a variable domain, wherein the variable domain comprises (i) an LC framework and a light chain containing a light chain complementarity determining region (LC-CDR) 1 with the amino acid sequence shown in SEQ ID NO: 11, LC-CDR 2 with the amino acid sequence shown in SEQ ID NO: 12, and LC-CDR 3 with the amino acid sequence shown in SEQ ID NO: 13; (ii) an LC framework and light chain complementarity determining region (LCCDR) 1 with the amino acid sequence shown in SEQ ID NO: 5, LC-CDR 2 with the amino acid sequence shown in SEQ ID NO: 6, and LC-CDR 3 with the amino acid the sequence shown in SEQ ID NO: 7; (iii) option (i) or (ii), wherein at least one of LC CDR 1, LCCDR 2 or LC-CDR 3 contains 1, 2 or 3 amino acid substitutions, insertions, deletions or combinations thereof; or (iv) option (i), (ii), or (iii), wherein the LC framework contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, insertions, deletions, or combinations thereof ; or (c) HC from (a) and LC from (b); wherein the antibody binds to the apple 3 domain of coagulation factor XI (FXI) and inhibits activation of FXI and/or factor XIa-mediated activation of factor IX.

In additional aspects or embodiments of the invention, the antibody of claim 18, wherein the HC constant domain comprises the amino acid sequence shown in SEQ ID NO: 16, 17, 18, or 19.

In additional aspects or embodiments, the antibody of claim 18 or 19, wherein the LC constant domain comprises the amino acid sequence shown in SEQ ID NO: 20.

The present invention further provides an antibody comprising a heavy chain with the amino acid sequence shown in SEQ ID NO: 33 and a light chain with the amino acid sequence shown in SEQ ID NO: 26.

The present invention further provides an antibody comprising a heavy chain with the amino acid sequence shown in SEQ ID NO: 35 and a light chain with the amino acid sequence shown in SEQ ID NO: 26.

The present invention further provides an antibody comprising a heavy chain with the amino acid sequence shown in SEQ ID NO: 45 and a light chain with the amino acid sequence shown in SEQ ID NO: 26.

The present invention further provides an antibody comprising a heavy chain with the amino acid sequence shown in SEQ ID NO: 47 and a light chain with the amino acid sequence shown in SEQ ID NO: 26.

The present invention further provides an antibody comprising a heavy chain with the amino acid sequence shown in SEQ ID NO: 49 and a light chain with the amino acid sequence shown in SEQ ID NO: 26.

The present invention further provides an antibody comprising a heavy chain with the amino acid sequence shown in SEQ ID NO: 51 and a light chain with the amino acid sequence shown in SEQ ID NO: 26.

The present invention further provides an antibody comprising a heavy chain with the amino acid sequence shown in SEQ ID NO: 59 and a light chain with the amino acid sequence shown in SEQ ID NO: 26.

The present invention further provides an antibody comprising a heavy chain with the amino acid sequence shown in SEQ ID NO: 61 and a light chain with the amino acid sequence shown in SEQ ID NO: 26.

The present invention further provides an antibody comprising a heavy chain with the amino acid sequence shown in SEQ ID NO: 63 and a light chain with the amino acid sequence shown in SEQ ID NO: 26.

The present invention further provides an antibody comprising a heavy chain with the amino acid sequence shown in SEQ ID NO: 69 and a light chain with the amino acid sequence shown in SEQ ID NO: 26.

The present invention further provides an antibody comprising a heavy chain with the amino acid sequence shown in SEQ ID NO: 33 and a light chain with the amino acid sequence shown in SEQ ID NO: 26.

The present invention further provides an antibody comprising a heavy chain with the amino acid sequence shown in SEQ ID NO: 71 and a light chain with the amino acid sequence shown in SEQ ID NO: 26.

The present invention further provides an antibody comprising a heavy chain with the amino acid sequence shown in SEQ ID NO: 73 and a light chain with the amino acid sequence shown in SEQ ID NO: 26.

The present invention further provides an antibody comprising a heavy chain with the amino acid sequence shown in SEQ ID NO: 75 and a light chain with the amino acid sequence shown in SEQ ID NO: 26.

The present invention further provides an antibody comprising a heavy chain with the amino acid sequence shown in SEQ ID NO: 39 and a light chain with the amino acid sequence shown in SEQ ID NO: 31.

The present invention further provides an antibody comprising a heavy chain with the amino acid sequence shown in SEQ ID NO: 41 and a light chain with the amino acid sequence shown in SEQ ID NO: 31.

The present invention further provides an antibody comprising a heavy chain with the amino acid sequence shown in SEQ ID NO: 43 and a light chain with the amino acid sequence shown in SEQ ID NO: 31.

The present invention further provides an antibody comprising a heavy chain with the amino acid sequence shown in SEQ ID NO: 53 and a light chain with the amino acid sequence shown in SEQ ID NO: 31.

The present invention further provides an antibody comprising a heavy chain with the amino acid sequence shown in SEQ ID NO: 55 and a light chain with the amino acid sequence shown in SEQ ID NO: 31.

The present invention further provides an antibody comprising a heavy chain with the amino acid sequence shown in SEQ ID NO: 57 and a light chain with the amino acid sequence shown in SEQ ID NO: 31.

The present invention further provides an antibody comprising a heavy chain with the amino acid sequence shown in SEQ ID NO: 65 and a light chain with the amino acid sequence shown in SEQ ID NO: 31.

The present invention further provides an antibody comprising a heavy chain with the amino acid sequence shown in SEQ ID NO: 67 and a light chain with the amino acid sequence shown in SEQ ID NO: 31.

The present invention further provides an antibody comprising a heavy chain with the amino acid sequence shown in SEQ ID NO: 69 and a light chain with the amino acid sequence shown in SEQ ID NO: 31.

The present invention further provides an antibody comprising a heavy chain with the amino acid sequence shown in SEQ ID NO: 77 and a light chain with the amino acid sequence shown in SEQ ID NO: 31.

The present invention further provides an antibody comprising a heavy chain with the amino acid sequence shown in SEQ ID NO: 79 and a light chain with the amino acid sequence shown in SEQ ID NO: 31.

The present invention further provides an antibody or antigen binding fragment that cross-blocks binding or competes for binding with an antibody comprising a heavy chain with the amino acid sequence shown in SEQ ID NO: 33, 35, 37, 45, 47, 49, 51, 59. 61, 63, 69, 71, 73 or 75 and a light chain with the amino acid sequence shown in SEQ ID NO:26; or an antibody comprising a heavy chain with the amino acid sequence shown in SEQ ID NO: 39, 41, 43, 53, 55, 57, 65, 67, 69, 77 or 79 and a light chain with the amino acid sequence shown in SEQ ID NO: 31 provided that the antibody or antigen binding fragment does not contain mouse or rat amino acid sequences.

In a further embodiment, the antibody or antigen binding fragment does not contain non-human amino acid sequences.

In a further embodiment, the antibody comprises (i) a human IgG1 constant domain or variant thereof, or a modified derivative thereof, or (ii) a human IgG4 constant domain or variant thereof, or a modified derivative thereof.

In a further embodiment, the IgG1 or IgG4 constant domain is a variant that contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, insertions, deletions, or combinations thereof.

In a further embodiment, the IgG1 or IgG4 constant domain is a variant that contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, insertions, deletions, or combinations thereof.

In a further embodiment, the IgG4 constant domain is a variant that comprises at least a replacement of serine at position 228 (EU numbering) or position 108, as shown herein, with a proline residue.

In a further embodiment, the IgG1 or IgG4 constant domain is a variant that at least lacks a lysine at the C-terminus.

In a further embodiment, the antibody or antigen binding fragment comprises variable domain sequences comprising a framework characteristic of human antibodies.

The present invention further provides an antibody or antigen binding fragment that cross-blocks binding or competes for binding with an antibody comprising a heavy chain with the amino acid sequence shown in SEQ ID NO: 33, 35, 37, 45, 47, 49, 51, 59. 61, 63, 69, 71, 73, or 75, and a light chain with the amino acid sequence shown in SEQ ID NO: 26; or an antibody comprising a heavy chain with the amino acid sequence shown in SEQ ID NO: 39, 41, 43, 53, 55, 57, 65, 67, 69, 77 or 79, and a light chain with the amino acid sequence shown in SEQ ID NO : 31.

In a further embodiment, the antibody or antigen binding fragment does not contain non-human amino acid sequences.

In a further embodiment, the antibody comprises (i) a human IgG1 constant domain or variant thereof, or a modified derivative thereof, or (ii) a human IgG4 constant domain or variant thereof, or a modified derivative thereof.

In a further embodiment, the IgG1 or IgG4 constant domain is a variant that contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, insertions, deletions, or combinations thereof.

In a further embodiment, the IgG1 or IgG4 constant domain is a variant that contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, insertions, deletions, or combinations thereof .

In a further embodiment, the IgG4 constant domain is a variant that contains at least a serine substitution at position 228 (EU numbering) or position

108, as shown herein, is a proline residue.

In a further embodiment, the IgG1 or IgG4 constant domain is a variant that at least lacks a lysine at the C-terminus.

In a further embodiment, the antibody or antigen binding fragment comprises variable domain sequences comprising a framework characteristic of human antibodies.

The present invention further provides an antibody or antigen binding fragment that binds to an epitope on coagulation factor XI (FXI) comprising the amino acid sequence YATRQFPSLEHRNICL (SEQ ID NO: 82) and the amino acid sequence HTQTGTPTRITKL (SEQ ID NO: 83) provided that the antibody or the antigen binding fragment does not contain mouse or rat amino acid sequences. In specific embodiments, epitope binding is determined by hydrogen-deuterium exchange mass spectrometry.

In a further embodiment, the antibody or antigen binding fragment does not contain non-human amino acid sequences.

In a further embodiment, the antibody comprises (i) a human IgG1 constant domain or variant thereof, or a modified derivative thereof, or (ii) a human IgG4 constant domain or variant thereof, or a modified derivative thereof.

In a further embodiment, the IgG1 or IgG4 constant domain is a variant that contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, insertions, deletions, or combinations thereof.

In a further embodiment, the IgG1 or IgG4 constant domain is a variant that contains at least 1, 2, 3 or 4 amino acid substitutions, insertions, deletions, or combinations thereof.

In a further embodiment, the IgG4 constant domain is a variant that comprises at least a replacement of serine at position 228 (EU numbering) or position 108, as shown herein, with a proline residue.

In a further embodiment, the IgG1 or IgG4 constant domain is a variant that at least lacks a lysine at the C-terminus.

In a further embodiment, the antibody or antigen binding fragment comprises variable domain sequences comprising a framework characteristic of human antibodies.

The present invention further provides an antibody or antigen binding fragment that binds to an epitope on coagulation factor XI (FXI) comprising the amino acid sequence YATRQFPSLEHRNICL (SEQ ID NO: 82) and the amino acid sequence HTQTGTPTRITKL (SEQ ID NO: 83) provided that the antibody contains i) a human IgG1 constant domain or variant thereof, or a modified derivative thereof, or (ii) a human IgG4 constant domain or variant thereof, or a modified derivative thereof. In specific embodiments, epitope binding is determined by hydrogen-deuterium exchange mass spectrometry.

In a further embodiment, the IgG1 or IgG4 constant domain is a variant that contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, insertions, deletions, or combinations thereof.

In a further embodiment, the IgG1 or IgG4 constant domain is a variant that contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, insertions, deletions, or combinations thereof.

In a further embodiment, the IgG4 constant domain is a variant that comprises at least a replacement of serine at position 228 (EU numbering) or position 108, as shown herein, with a proline residue.

In a further embodiment, the IgG1 or IgG4 constant domain is a variant that at least lacks a lysine at the C-terminus.

In a further embodiment, the antibody or antigen binding fragment comprises variable domain sequences comprising a framework characteristic of human antibodies.

The present invention further relates to an isolated nucleic acid molecule encoding a light chain variable domain or a heavy chain variable domain of any of the above antibodies or antigen binding fragments.

The present invention further provides a humanized antibody or antigen binding fragment that binds to an epitope on coagulation factor XI (FXI) comprising the amino acid sequence YATRQFPSLEHRNICL (SEQ ID NO: 82) and the amino acid sequence HTQTGTPTRITKL (SEQ ID NO: 83) provided that the antibody contains i) a human IgG1 constant domain or variant thereof, or a modified derivative thereof, or (ii) a human IgG4 constant domain or variant thereof, or a modified derivative thereof. In specific embodiments, binding to an epitope is determined by hydrogen-deuterium mass spectrometry

- 11 043303 exchange.

In a further embodiment, the IgG1 or IgG4 constant domain is a variant that contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, insertions, deletions, or combinations thereof.

In a further embodiment, the IgG1 or IgG4 constant domain is a variant that contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, insertions, deletions, or combinations thereof.

In a further embodiment, the IgG4 constant domain is a variant that comprises at least a replacement of serine at position 228 (EU numbering) or position 108, as shown herein, with a proline residue.

In a further embodiment, the IgG1 or IgG4 constant domain is a variant that at least lacks a lysine at the C-terminus.

In a further embodiment, the antibody or antigen binding fragment comprises variable domain sequences comprising a framework characteristic of human antibodies.

The present invention further relates to an isolated nucleic acid molecule encoding a light chain variable domain or a heavy chain variable domain of any of the above antibodies or antigen binding fragments.

The present invention further provides a composition comprising an antibody or antigen binding fragment of any of the above antibodies or antigen binding fragments and a pharmaceutically acceptable carrier or diluent.

The present invention further provides a method of treating a thromboembolic disorder or disease in an individual, comprising administering to the individual an effective amount of any antibody or antigen binding fragment of the above antibodies or antigen binding fragments.

The present invention further provides a method of treating a thromboembolic disorder or disease in an individual, comprising administering to the individual in need thereof an effective amount of any antibody or antigen binding fragment of the above antibodies or antigen binding fragments.

The present invention further relates to the use of any of the above antibodies or antigen binding fragments for the preparation of a medicament for the treatment of a thromboembolic disorder or disease.

The present invention further relates to any of the above antibodies or antigen binding fragments for the treatment of a thromboembolic disorder or disease.

The present invention further relates to a method for producing an antibody or antigen binding fragment comprising (i) a heavy chain with a constant domain and a variable domain, wherein the variable domain comprises a heavy chain containing a heavy chain complementarity determining region (HC-CDR) 1 with the amino acid sequence shown in SEQ ID NO: 1, HC-CDR 2 with the amino acid sequence shown in SEQ ID NO: 2, and HC-CDR 3 with the amino acid sequence shown in SEQ ID NO: 3 or 4; and (ii) a light chain with a constant domain and a variable domain, wherein the variable domain comprises a light chain complementarity determining region (LC-CDR) 1 with the amino acid sequence shown in SEQ ID NO: 5, LC-CDR 2 with the amino acid sequence shown in SEQ ID NO: 6, and LC-CDR 3 with the amino acid sequence shown in SEQ ID NO: 7, a method comprising creating a host cell containing a nucleic acid molecule encoding a heavy chain and a nucleic acid molecule encoding a light chain; and culturing the host cell under conditions and for a period of time sufficient to produce an antibody or antigen-binding fragment.

In additional aspects or embodiments of the invention, the antibody comprises a heavy chain constant domain of an IgG1, IgG2, IgG3, or IgG4 isotype.

In additional aspects or embodiments of the invention, the antibody comprises a heavy chain constant domain of the IgG4 isotype.

In additional aspects or embodiments of the invention, the antibody comprises a heavy chain constant domain comprising the amino acid sequence shown in SEQ ID NO: 16, 17, 18, or 19.

In additional aspects or embodiments of the invention, the light chain comprises a human kappa light chain or a human lambda light chain.

In further aspects or embodiments of the invention, the antibody comprises a light chain constant domain comprising the amino acid sequence shown in SEQ ID NO: 20.

In additional aspects or embodiments of the invention, the host cell is a Chinese hamster ovary cell or a human embryonic kidney 293 cell.

In additional aspects or embodiments of the invention, the host cell is a yeast or filamentous fungus cell.

- 12 043303

The present invention further relates to a method for producing an antibody or antigen binding fragment comprising (i) a heavy chain with a constant domain and a variable domain, wherein the variable domain comprises a heavy chain containing a heavy chain complementarity determining region (HC-CDR) 1 with the amino acid sequence shown in SEQ ID NO: 1, HC-CDR 2 with the amino acid sequence shown in SEQ ID NO: 2, and HC-CDR 3 with the amino acid sequence shown in SEQ ID NO: 3 or 4; and (ii) a light chain with a constant domain and a variable domain, wherein the variable domain comprises a light chain complementarity determining region (LC-CDR) 1 with the amino acid sequence shown in SEQ ID NO: 5, LC-CDR 2 with the amino acid sequence shown in SEQ ID NO: 6, and LC-CDR 3 with the amino acid sequence shown in SEQ ID NO: 7, a method comprising creating a host cell containing a nucleic acid molecule encoding a heavy chain and a nucleic acid molecule encoding a light chain; and culturing the host cell under conditions and for a period of time sufficient to produce the antibody or antigen binding fragment.

in further aspects or embodiments, the heavy chain stant domain of an IgG1, IgG2, IgG3, or IgG4 isotype.

In additional aspects or embodiments, the heavy chain constant domain of the IgG4 isotype.

In additional aspects or embodiments of the invention, the antibody comprises a coni-invention; the antibody comprises a con-antibody of the con-invention comprises a heavy chain constant domain comprising the amino acid sequence shown in SEQ ID NO: 16, 17, 18, or 19.

In additional aspects or embodiments of the invention, the light chain comprises a human kappa light chain or a human lambda light chain.

In further aspects or embodiments of the invention, the antibody comprises a light chain constant domain comprising the amino acid sequence shown in SEQ ID NO: 20.

In additional aspects or embodiments of the invention, the host cell is a Chinese hamster ovary cell or a human embryonic kidney 293 cell.

In additional aspects or embodiments of the invention, the host cell is a yeast or filamentous fungus cell.

A method of producing an antibody or antigen binding fragment containing (i) a heavy chain variable domain containing a heavy chain complementarity determining region (HC-CDR) 1 with the amino acid sequence shown in SEQ ID NO: 1, HC-CDR 2 with the amino acid sequence shown in SEQ ID NO: 2, and HC-CDR 3 with the amino acid sequence shown in SEQ ID NO: 3 or 4, or HC-CDR 1 with the amino acid sequence shown in SEQ ID NO: 8, HC-CDR 2 with the amino acid sequence, shown in SEQ ID NO: 9, and HCCDR 3 with the amino acid sequence shown in SEQ ID NO: 10; and (ii) a light chain variable domain comprising a light chain complementarity determining region (LC-CDR) 1 with the amino acid sequence shown in SEQ ID NO: 5, LC-CDR 2 with the amino acid sequence shown in SEQ ID NO: 6, and LC-CDR 3 with the amino acid sequence shown in SEQ ID NO: 7, or LC-CDR 1 with the amino acid sequence shown in SEQ ID NO: 11, LC-CDR 2 with the amino acid sequence shown in SEQ ID NO: 12, and LC-CDR 3 with the amino acid sequence shown in SEQ ID NO: 13, a method comprising creating a host cell containing a nucleic acid molecule encoding a heavy chain and a nucleic acid molecule encoding a light chain; and culturing the host cell under conditions and for a period of time sufficient to produce an antibody or antigen-binding fragment.

In additional aspects or embodiments, the heavy chain stant domain is of an IgG1, IgG2, IgG3, or IgG4 isotype.

In additional aspects or embodiments, the heavy chain constant domain of the IgG4 isotype.

In additional aspects or embodiments of the invention, the antibody comprises a coninvention; the antibody comprises a conantibody of the coninvention comprises a heavy chain constant domain comprising the amino acid sequence shown in SEQ ID NO: 16, 17, 18, or 19.

In additional aspects or embodiments of the invention, the light chain comprises a human kappa light chain or a human lambda light chain.

In further aspects or embodiments of the invention, the antibody comprises a light chain constant domain comprising the amino acid sequence shown in SEQ ID NO: 20.

In additional aspects or embodiments of the invention, the host cell is a Chinese hamster ovary cell or a human embryonic kidney 293 cell.

In additional aspects or embodiments of the invention, the host cell is a yeast or filamentous fungus cell.

The present invention further relates to a composition containing any of the following

- 13 043303 higher antibodies and pharmaceutically acceptable carrier. In specific embodiments, the composition contains a mixture of antibodies containing a heavy chain with a C-terminal lysine and antibodies containing a heavy chain without a C-terminal lysine. In specific embodiments, the composition comprises an antibody described herein, wherein the predominant form of the antibody comprises a C-terminal lysine heavy chain. In specific embodiments, the composition contains an antibody described herein, wherein the predominant form of the antibody contains a heavy chain lacking the C-terminal lysine. In specific embodiments, the composition contains an antibody described herein, wherein approximately 100% of the antibodies in the composition contain a heavy chain without a C-terminal lysine.

Definitions

As used herein, antibody refers to both the entire immunoglobulin, including recombinantly produced forms, and includes any form of the antibody that exhibits the desired biological activity. Thus, it is applied in the broadest sense and specifically includes, as non-limiting examples, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized, fully human antibodies, biparatope antibodies, and chimeric antibodies. Parent antibodies are antibodies produced by presenting an antigen to the immune system before modifying the antibodies for a given use, such as humanizing the antibody for use as a therapeutic antibody in humans.

An antibody in one embodiment refers to a glycoprotein containing at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain consists of a heavy chain variable region (abbreviated herein as _VH ) and a heavy chain constant region. In certain natural antibodies IgG, IgD and IgA, the heavy chain constant region consists of three domains, CH1, CH2 and CH3. In certain natural antibodies, each light chain consists of a light chain variable region (abbreviated herein as V _L ) and a light chain constant region. The light chain constant region consists of a single CL domain. The VH and _VL regions can be further divided into regions of hypervariability called complementarity determining regions (CDRs), separated by more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, located from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with the antigen. Antibody constant regions can mediate the binding of immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.

Typically, the basic structural unit of an antibody contains a tetramer. Each tetramer contains two identical pairs of polypeptide chains, each pair with one light chain (approximately 25 kDa) and one heavy chain (approximately 50-70 kDa). The amino-terminal portion of each chain includes a variable region of approximately 100 to 110 or more amino acids initially responsible for antigen recognition. The carboxy-terminal portion of the heavy chain may define a constant region initially responsible for effector function. Generally, human light chains are classified as kappa and lambda light chains. In addition, human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and the antibody isotype is defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a J region of approximately 12 or more amino acids, and the heavy chain also includes a D region of approximately 10 more amino acids. See mainly Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)).

The antibody heavy chain may or may not contain a terminal lysine (K), or a terminal glycine and lysine (GK). Thus, in specific embodiments, antibodies herein containing a heavy chain constant region amino acid sequence shown herein that does not contain a terminal lysine and ends with a glycine residue further include embodiments that also lack a terminal glycine residue. This occurs because the terminal lysine and sometimes the glycine and lysine together are cleaved off during antibody expression.

As used herein, an antigen binding fragment refers to antibody fragments, i.e. antibody fragments that retain the ability to specifically bind to an antigen to which the full-length antibody binds, for example, fragments that retain one or more CDR regions. Examples of antibody binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; single chain antibody molecules, for example sc-Fv; nanobodies and polyspecific antibodies formed from antibody fragments.

As used herein, a Fab fragment consists of one light chain and the _CH 1 and variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. The Fab fragment may be a papain cleavage product of the antibody.

As used herein, the Fab' fragment contains one light chain and a portion or fragment of one heavy chain that contains a VH domain and a _CH 1 domain and also a region between the _CH 1 and CH 2 domains, such that an intrachain disulfide moiety can be formed. linkage between the two heavy chains of the two Fab' fragments to form the F(ab') ₂ molecule.

As used herein, the F(ab') ₂ fragment contains two light chains and two heavy chains, including a VH domain and part of the constant region between the CH1 and CH2 domains, such that an intrachain disulfide bond is formed between the two heavy chains. The F(ab')2 fragment thus consists of two Fab' fragments that are held together by a disulfide bond between the two heavy chains. The F(ab') ₂ fragment may be a product of the cleavage of the antibody by pepsin.

As used herein, the Fv region contains heavy and light chain variable regions, but does not contain constant regions.

These and other potential constructs are described in Chan & Carter (2010) Nat. Rev. Immunol. 10:301. These antibody fragments are prepared using conventional methods known to those skilled in the art, and the fragments are selected for their abilities in the same manner as intact antibodies. Antigen-binding moieties can be produced by recombinant DNA methods, or by enzymatic or chemical digestion of intact immunoglobulins.

As used herein, the Fc region contains two heavy chain fragments comprising the CH1 and CH2 domains of the antibody. The two heavy chain moieties are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains.

As used herein, a diabody refers to a small antibody fragment with two antigen-binding regions, the fragments containing a heavy chain variable domain (VH) linked to a light chain variable domain ( _VL ) in a single polypeptide chain (VH-VL or _{VL -VH} ). Using a linker that is too short to allow pairing between two domains on one strand, the domains are forced to pair with complementary domains on the other strand and create two antigen-binding sites. Diabodies are described more fully, for example in EP 404097; WO 93/11161; and Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448. For a review of engineered antibody variants, see generally Holliger and Hudson (2005) Nat. Biotechnol. 23:11261136.

As used herein, a bispecific antibody is an artificial hybrid antibody with two different heavy/light chain pairs, and thus two different binding sites. For example, the bispecific antibody may comprise a first heavy/light chain pair with one heavy and one light chain from the first antibody containing at least six CDRs from the antibody aFXI-13654p, aFXI-13716p, or aFXI-13716, or embodiments, wherein one or more of the six CDRs have one, two or three amino acid substitutions, insertions, deletions, or combinations thereof, together with a second heavy/light chain pair with one heavy and one light chain from a second antibody with specificity for an antigen of interest other than FXI . Bispecific antibodies can be produced by a number of methods, including hybridoma fusion or ligation of Fab' fragments. See, for example, Songsivilai, et al., (1990) Clin. Exp. Immunol. 79: 315-321, Kostelny, et al., (1992) J Immunol. 148:1547–1553.

In addition, bispecific antibodies can be formed as diabodies (Holliger, et al., (1993) PNAS USA 90:6444-6448) or as Janusins (Traunecker et al., (1991) EMBO J. 10:3655-3659 and Traunecker , et al., (1992) Int. J. Cancer Suppl. 7:51-52).

As used herein, isolated antibodies or antigen-binding fragments thereof are at least partially free from other biological molecules from the cells or cell cultures in which they are obtained. Such biological molecules include nucleic acids, proteins, lipids, carbohydrates, or other material such as cell debris and growth media. The isolated antibody or antigen binding fragment may further be at least partially free from components of the expression system, such as biological molecules from the host cell or its growth medium. Generally, the term isolated is not intended to refer to the complete absence of such biological molecules or the absence of water, buffers or salts or components of a pharmaceutical formulation that includes antibodies or fragments.

As used herein, a monoclonal antibody refers to a population of essentially homogeneous antibodies, i.e., antibody molecules comprising a population identical in amino acid sequence, excluding possible naturally occurring mutations that may be present in minute quantities. In contrast, conventional (polyclonal) antibody preparations typically include many different antibodies with different amino acid sequences in the variable domains, which are often specific for different epitopes. The modifier monoclonal indicates the characteristic of the antibody as being derived from an essentially homogeneous population

- 15 043303 antibodies, and is not considered to require obtaining the antibody by any particular method. For example, monoclonal antibodies for use in accordance with the present invention can be produced by the hybridoma method first described by Kohler et al. (1975) Nature 256: 495, or can be produced by recombinant DNA methods (see, for example, US patent No. 4816567). Monoclonal antibodies can also be isolated from phage antibody libraries using the methods described in Clackson et al. (1991) Nature 352: 624-628 and Marks et al. (1991) J. Mol. Biol. 222: 581-597, for example, see also Presta (2005) J. Allergy Clin. Immunol. 116:731.

As used herein, a chimeric antibody is an antibody having a variable domain from a first antibody and a constant domain from a second antibody, wherein (i) the first and second antibodies are from different species (US Pat. No. 4,816,567 and Morrison et al., (1984) Proc. Natl. Acad. Sci. USA 81: 6851-6855) or (ii) first and second antibodies from different isotypes, for example, the variable domain from an IgG1 antibody and constant domains from an IgG4 antibody, for example aFXI-134 65p-IgG4 (S228P) . In one aspect, the variable domains are derived from a human antibody (parent antibody) and the constant domain sequences are derived from a non-human antibody (eg, mouse, rat, dog, monkey, gorilla, horse). In another aspect, the variable domains are derived from a non-human antibody (parent antibody) (eg, mouse, rat, dog, monkey, gorilla, horse) and the constant domain sequences are derived from a human antibody. In a further aspect, the variable domains are derived from a human IgG1 antibody (the parent antibody) and the constant domain sequences are derived from a human IgG4 antibody.

As used herein, a humanized antibody refers to forms of antibodies that contain sequences from human and non-human (eg, murine, rat) antibodies. In general, a humanized antibody will contain all of at least one, and typically two, variable domains in which the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the framework regions (FRs) are human immunoglobulin sequences. The humanized antibody may optionally contain at least a portion of a human immunoglobulin constant region (Fc).

As used herein, a fully human antibody refers to an antibody that contains human immunoglobulin amino acid sequences or variant sequences thereof that contain mutations introduced recombinantly to create a fully human antibody with modified function or potency compared to an antibody in which there are no specified mutations. A fully human antibody does not contain non-human immunoglobulin amino acid sequences, for example, constant domains and variable domains, including CDRs, contain human sequences other than those derived from the mutations described above. A fully human antibody may include amino acid sequences of antibodies or immunoglobulins obtained from a library of fully human antibodies, the diversity in the library being obtained in silico (See, for example, US Pat. No. 8,877,688 or 8,691,730). A fully human antibody includes those antibodies produced in a non-human body, for example, a fully human antibody may contain murine carbohydrate chains if produced in a mouse, in a mouse cell, or in a hybridoma that is derived from a mouse cell. Likewise, mouse antibody or murine antibody refers to an antibody that contains only mouse immunoglobulin sequences. Alternatively, the fully human antibody may contain rat carbohydrate chains if produced by a rat, in rat cells, or in a hybridoma that is derived from rat cells. Similarly, a rat antibody refers to an antibody that contains only rat immunoglobulin sequences.

As used herein, non-human amino acid sequences in relation to antibodies or immunoglobulins refers to an amino acid sequence that is representative of the amino acid sequence of a non-human mammal. The term does not include the amino acid sequences of antibodies or immunoglobulins obtained from a library of fully human antibodies, the diversity in the library being obtained in silico (See, for example, US Pat. No. 8877688 or 8691730).

As used herein, effector functions refer to those biological activities associated with the Fc region of an antibody, which vary depending on the antibody isotype. Examples of antibody effector functions include: Clq binding and complement-mediated cytotoxicity (CDC), Fc receptor binding, antibody-dependent cellular cytotoxicity (ADCC), phagocytosis, negative regulation of cell surface receptors (eg, B cell receptor), and B cell activation.

The variable regions of each light/heavy chain pair form the binding site of the antibody. Thus, basically, an intact antibody has two binding sites. With the exception of bifunctional or bispecific antibodies, the two binding sites are usually the same.

Typically, the variable domains of both the heavy and light chains contain three hypervariable regions, also called complementarity determining regions (CDRs), located

- 16 043303 within relatively conserved framework regions (FR). CDRs are typically aligned with framework regions to allow binding to a specific epitope. Generally, from N-terminus to C-terminus, the light and heavy chain variable domains contain FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain generally follows the definitions from Sequences of Proteins of Immunological Interest, Kabat, et al.; National Institutes of Health, Bethesda, Md.; 5 ^th ed.; NIH Publ. No. 91-3242 (1991); Kabat (1978) Adv. Prot. Chem. 32:1-75; Rabat, et al., (1977) J. Biol. Chem. 252:6609-6616; Chothia, et al., (1987) J Mol. Biol. 196:901-917 or Chothia et al., (1989) Nature 342: 878-883.

As used herein, a hypervariable region refers to the amino acid residues of an antibody that are responsible for binding to an antigen. The hypervariable region contains amino acid residues from the complementarity determining region or CDR (ie CDRL1, CDRL2 and CDRL3 in the light chain variable domain and CDRH1, CDRH2 and CDRH3 in the heavy chain variable domain). See Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (determination of antibody CDR regions by sequence); see also Chothia and Lesk (1987) J. Mol. Biol. 196: 901-917 (identification of antibody CDR regions by structure).

As used herein, framework or FR residues refer to variable domain residues other than hypervariable region residues, defined herein as CDR residues.

As used herein, conservatively modified variants or conservative substitution refers to the replacement of amino acids with other amino acids with similar characteristics (e.g., charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.) such that substitutions are often made without changing the biological activity of the protein. Those skilled in the art will know that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, for example, Watson et al. (1987) Molecular Biology of Gene, The Benjamin/Cummings Pub. Co. , p. 224 (4th Ed.)).

In addition, substitutions with structurally or functionally similar amino acids are less likely to interfere with biological activity. Examples of conservative substitutions are presented in the table below.

Original balance

Conservative Initial

Conservative replacement remainder replacement

Ala (A)

Arg(R)

Asn(N)

Asp (D)

Cys(C)

Gln(Q)

Glu(E)

Gly (G)

His(H)

Gly; Ser

Lys; His

Gln; His

Glu; Asn

Ser; Ala

Leu (L)

Lys (K)

Met(M)

Phe(F)

Pro (P)

Ser (S)

Asp; Gln

Thr (T)

Trp(W)

Asn; Gln

Tyr (Y) lie; Vai

Arg; His

Leu; lie; Tyr

Tyr; Met; Leu

Tyr; Phe

Trp; Phe

Leu; Vai

Vai (V)

Ile; Leu

As used herein, the term epitope or antigenic determinant refers to a region on an antigen (eg, FXI) to which an immunoglobulin or antibody specifically binds. Epitopes within protein antigens can be formed from contiguous amino acids (typically a linear epitope) or non-contiguous amino acids placed adjacent by tertiary protein folding (typically a conformational epitope). Epitopes formed by contiguous amino acids are generally, but not always, retained when exposed to denaturing solvents, while epitopes formed by tertiary folding tend to disappear when exposed to denaturing solvents. An epitope typically comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a unique spatial conformation. Methods for determining which epitopes bind to an antibody (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays, where overlapping or adjacent peptides (eg, from FXI) are tested for reactivity with the specified antibody (eg, anti-FXI antibody). Methods for determining the spatial conformation of epitopes include methods in the art and methods described herein, for example, X-ray crystallography, two-dimensional nuclear magnetic resonance, and HDX-MS (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996)).

The term epitope mapping refers to a method of identifying molecular determinants on an antigen involved in antibody-antigen recognition.

The term binds to the same epitope in relation to two or more antibodies means that the antibodies bind to the same segment of amino acid residues specified

- 17 043303 in a different way. Methods for determining whether antibodies with the same epitope on FXI bind to the antibodies described herein include, for example, epitope mapping methods such as X-ray crystal analysis of antigen:antibody complexes, which provides atomic resolution of the epitope, and mass hydrogen-deuterium exchange spectrometry (HDXMS). Other methods that monitor antibody binding to fragments of the antigen (eg, proteolytic fragments) or to mutated variations of the antigen, where lack of binding due to modification of an amino acid residue within the antigen sequence are often taken to indicate a component of the epitope (eg, alanine scanning mutagenesis, Cunningham & Wells (1985) Science 244:1081). In addition, computational combinatorics methods can also be used to map epitopes. These methods rely on the ability of the antibody of interest to affinity isolate specific short peptides from phage display-based combinatorial peptide libraries.

Antibodies that compete with another antibody for binding to a target, such as FXI, refer to antibodies that inhibit (partially or completely) the binding of another antibody to the target. Whether two antibodies compete with each other for binding to a target, i.e., whether and to what extent one antibody inhibits the binding of another antibody to a target, can be determined using competition experiments. In certain embodiments, the antibody competes with another antibody and inhibits binding of the antibody to its target by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. The level of inhibition or competition may vary depending on which antibody is the blocking antibody (ie, the cold antibody that is incubated first with the target). Competitive assays can be performed as described, for example, in Ed Harlow and David Lane, Cold Spring Harb Protoc; 2006; doi:10.1101/pdb.prot4277 or in Chapter 11 Using Antibodies by Ed Harlow and David Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA 1999. Competitive antibodies bind to the same epitope, an overlapping epitope, or adjacent epitopes (eg, as demonstrated by steric hindrance). Other competitive binding assays include solid-phase direct and indirect radioimmunoassay (RIA), solid-phase direct and indirect enzyme immunoassay (EIA), competitive sandwich assay (see Stahli et al., Methods in Enzymology 9:242 (1983)); solid phase direct biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614 (1986)); solid-phase direct labeled assay, solid-phase direct labeled sandwich assay (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); solid-phase direct labeled RIA using the I-125 label (see Morel et al., Mol. Immunol. 25(1):7 (1988)); solid phase direct biotinavidin EIA (Cheung et al., Virology 176:546 (1990)); and direct labeled RIA (Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)).

As used herein, specifically binds to an antigen or molecule, such as FXI, refers to the preferential association of an antibody or other ligand, in whole or in part, with FXI rather than with other molecules, particularly molecules found in the blood or human serum. Antibodies typically bind specifically to the recognized antigen with high affinity, which is reflected by a dissociation constant (KD) of 10 ^-7 to 10 ^-11 M or less. Any KD greater than about 10 ^-6 M is generally considered to indicate nonspecific binding. As used herein, an antibody that specifically binds an antigen refers to an antibody that binds to an antigen and substantially identical antigens with high affinity, i.e. with a KD of 10 ^-7 M or less, in certain embodiments with a KD of 10 ^-8 M or less, or 5x10' ⁹ M or less, or between 10 ^-8 M and 10 ^-11 M or less, but does not bind with high affinity with unrelated antigens. Binding kinetics can be determined by surface plasmon resonance as described in Example 1 herein.

An antigen is substantially identical to a given antigen if it exhibits a high degree of amino acid sequence identity to the antigen, for example, if it exhibits at least 80%, at least 90%, at least 95%, at least 97%, or at least 99% or greater amino acid sequence identity with the amino acid sequence of a given antigen. As an example, an antibody that specifically binds to human FXI may also cross-react with FXI from certain non-human primate species (eg, cynomolgus monkey), but may not cross-react with FXI from other species, or with an antigen other than FXI.

As used herein, an isolated nucleic acid molecule means genomic DNA or RNA, mRNA, cDNA, or synthetic origin, or any combination thereof, that is not associated with a polynucleotide or part thereof in which the isolated polynucleotide occurs naturally, or is associated with polynucleotide to which it is not associated in nature. For purposes of the present description, it should be understood that the nucleic acid molecule containing a particular nucleotide sequence does not include intact chromosomes. Isolated nucleic acid molecules containing defined nucleic acid sequences may include, in addition to the defined sequences, up to ten or even up to twenty coding sequences.

- 18 043303 more proteins or parts or fragments thereof, or may include operably linked regulatory sequences that control the expression of the coding region of said nucleic acid sequences, and/or may include vector sequences.

As used herein, treat or treat refers to the administration of an internally or externally therapeutic agent, such as a composition containing any of the antibodies or antigen-binding fragments thereof of the present invention, to an individual or patient with one or more symptoms of a disease or suspected of having a disease, for which the agent has therapeutic activity or prophylactic activity. Typically, the agent is administered in an amount effective to relieve one or more symptoms of a disease in the individual or population being treated, or by causing regression or inhibiting the progression of such symptom(s) to any clinically measurable degree. The amount of therapeutic agent that is effective in alleviating any particular disease symptom may vary according to factors such as the disease state, the age, and weight of the patient, and the ability of the drug to produce the desired response in the individual. Whether a disease syndrome will improve can be assessed using any clinical measure typically used by physicians or other qualified health care professionals to assess the severity or progression status of that symptom. The term further includes delaying the development of symptoms associated with a disorder and/or reducing the severity of symptoms of such a disorder. The terms further include improvement of existing uncontrolled or unwanted symptoms, prevention of additional symptoms, and improvement or prevention of the underlying causes of such symptoms. Thus, the terms mean that a beneficial outcome has been achieved in a person or animal with a disorder, disease or symptom, or with the potential to develop such a disorder, disease or symptom.

As used herein, treatment, when applied to a human or veterinary subject, refers to therapeutic treatment as well as diagnostic applications. Treatment, as applied to a human or veterinary subject, involves contacting the antibodies or antigen-binding fragments of the present invention with the human or animal.

As used herein, a therapeutically effective amount refers to an amount of a particular substance sufficient to achieve the desired effect in the individual being treated. For example, it may be the amount required to inhibit FXI activation or the amount required to inhibit coagulation for at least 192 to 288 hours, as determined by aPTT assay. When administered to an individual, a dose will generally be used that achieves target tissue concentrations that have been shown to achieve the desired effect in vitro.

As used herein, thrombosis refers to the formation or presence of a clot (also called a thrombus) within a blood vessel, obstructing the flow of blood through the circulatory system. Thrombosis is usually caused by disturbances in the composition of the blood, the quality of the vascular wall and/or the nature of the blood flow. Clot formation is often caused by damage to the vascular wall (such as injury or infection) and slowing or stopping of blood flow past the point of injury. In some cases, clotting disorders cause thrombosis.

As used herein, non-impaired hemostasis means that the individual or patient experiences little or no detectable bleeding following administration of an antibody or antibody fragment described herein to the individual or patient. When the target is factor XI, inhibition of the conversion of factor XI to factor XIa or the activation of factor IX by factor XIa inhibits coagulation and associated thrombosis without bleeding. In contrast, inhibition of factor XI conversion or activity inhibits clotting but also causes bleeding or increases the risk of bleeding.

Brief description of drawings

Fig. 1A and 1B show the coagulation cascade, FXI, anti-FXI mAbs, and four new oral anticoagulants (NOACs). Fig. 1A is a figure showing FXI in the coagulation cascade (which consists of the intrinsic and extrinsic pathways). An mAb targeting FXI may exert functional neutralization by blocking the activation of FXI by XIIa and/or thrombin, or the effect of FXIa on FIX. The antibodies herein can exert dual blockade on FXIa-mediated activation of FIX, and the conversion of FXI to FXIa mediated by at least FXIIa. Four NOACs (rivaroxaban, apixaban, edoxaban, dabigatran) targeting either FXa or thrombin are indicated. Fig. 1B shows the domain structure of FXI. FXI is a dimer consisting of identical 80 kDa subunits, and each subunit, starting at the N terminus, consists of four apple domains (1, 2, 3, and 4) and a catalytic domain (CAT). The antibodies described herein bind to the apple 3 domain.

In fig. Figure 2 shows the structure of factor XI and apple 3 domain with peptides protected from deuteration by identified antibodies to FXI from the aFXI-18611 and aFXI18623p family. Shown is arginine residue 184, a critical residue in the outer FIX binding site. Peptides

- 19 043303 in the Apple 3 domain with no difference in deuteration are shown in light gray. Peptides with missing data are shown in dark grey. The catalytic domain is not shown.

Fig. 3A and 3B show a heat map of the differences in deuterium labeling of FXI amino acid residues bound to the anti-FXI antibodies aFXI-18611 IgG4 HC (S228P) (E1) (L105)/LC kappa and aFXI18623p IgG4 HC (S228P) (Q1)/LC kappa, respectively.

Fig. 4A, 4B, and 4C show the amino acid sequence of the HC and LC domains of the aFXI 18611p and aFXI 18611 family antibodies. The heavy chain and light chain CDRs are identified as HC-CDR1, HC-CDR2, HC-CDR3, LC-CDR1, LC-CDR2, and LC -CDR3 respectively.

Fig. 5A and 5B show the amino acid sequence of the HC and LC domains of the aFXI 18623p family antibodies. The heavy chain and light chain CDRs are identified as HC-CDR1, HC-CDR2, HCCDR3, LC-CDR1, LC-CDR2, and LC-CDR3, respectively.

In fig. 6 shows the results of the analysis of activated partial thromboplatin time (APTT) aFXI-18611 IgG4 NS (S228P) (E1) (L105)/LC kappa (A) and aFXI-18623p IgG4 NS (S228P) (Q1)/LC kappa (B) in human plasma, expressed as a % increase from baseline.

In fig. 7 presents the results of the analysis of activated partial thromboplatin time (aPTT) aFXI-18611 IgG4 NS (S228P) (E1) (L105)/LC kappa (A) and aFXI-18623p IgG4 NS (S228P) (Q1)/LC kappa (B) in cynomolgus monkey plasma, expressed as % increase from baseline.

In fig. 8 shows the results of the analysis of activated partial thromboplatin time (aPTT) aFXI-18611 IgG4 NS (S228P) (E1) (L105)/LC kappa (A) and aFXI-18623p IgG4 NS (S228P) (Q1)/LC kappa (B) in plasma from rhesus monkeys, expressed as a % increase from baseline.

In fig. 9 shows a comparison of aPTT results for aFXI-18611 IgG4 HC (S228P) (E1) (L105)/LC kappa in human, cynomolgus and rhesus monkey plasma, expressed as % increase from baseline.

In fig. 10 shows a comparison of aPTT results for aFXI-18623p IgG4 HC (S228P) (Q1)/LC kappa in human, cynomolgus and rhesus monkey plasma, expressed as % increase from baseline.

In fig. 11 shows BIAcore sensorgrams that show the binding kinetics of aFXI18623p IgG4 HC (S228P) (E1)/LC Kappa to human, cynomolgus and rhesus FXI and to other human and NHP coagulation cascade proteins.

In fig. 12 shows BIAcore sensorgrams that show the binding kinetics of aFXI18623p IgG4 HC (S228P) (Q1)/LC Kappa to human, cynomolgus and rhesus FXI and to other human and NHP coagulation cascade proteins.

In fig. Figure 13 shows a schematic test model of an arteriovenous shunt (AV) in a cynomolgus monkey.

Anesthetized monkeys with pre-installed catheters were injected into the femoral artery and vein with vehicle or aFXI-18623p IgG4 HC (S228P) (E1)/LC Kappa (antibody) at 0.011.0 mg/kg by intravenous bolus (test drug administration). The AV shunt was inserted as described in the text (AV shunt insertion). Blood flowed through the AV shunt for 40 minutes. Contact between the blood and the silk thread suspended inside the tube caused a clot to form. Clots were weighed as described in the text. Blood samples were obtained to measure circulating antibody, APTT, and RT levels (asterisks).

Fig. 14A-14D show the effects of aFXI-18623p IgG4 HC (S228P) (E1)/LC Kappa (antibody) on AV shunt clot formation, APTT and RT in a cynomolgus monkey AV shunt model. Fig. 14A, Clot mass measured after two consecutive AV shunts in the same animal. Animals were treated with vehicle during the first shunt (shunt #1), followed by antibody (0.01-1.0 mg/kg IV) as indicated during the second shunt (shunt #2). Increasing doses of the antibody led to the formation of smaller clots. The percentage inhibition of clot mass (Fig. 14B) and the percentage change in aPTT (Fig. 14C) increased with increasing plasma antibody concentration. In contrast, PT (Fig. 14D) remained relatively unchanged at all antibody concentrations.

In fig. Figure 15 shows a diagram of the template model of bleeding time in the cynomolgus monkey. Patterned bleeding times at the buccal mucosa (inner lip), finger pad, and tail tip were determined in anesthetized cynomolgus monkeys at baseline (pre-treatment) and after administration of Treatment#1 (vehicle) and Treatment#2 (vehicle or aFXI-18623p IgG4 HC). S228P) (E1)/LC Kappa, 10 mg/kg i.v.). Blood samples to measure circulating levels of aFXI-18623p IgG4 HC (S228P) (E1)/LC Kappa, APTT, and RT were collected as indicated.

Fig. 16A-16F show the effects of aFXI-18623p IgG4 HC (S228P) (E1)/LC Kappa on patterned bleeding time measured in cynomolgus monkeys. Pattern bleeding time was measured in the buccal mucosa (FIGS. 16A, 16D), fingertip (FIGS. 16B, 16E) and tip of the tail (FIGS. 16C, 16F). The effects of treatment (aFXI-18623p IgG4 HC (S228P) (E1)/LC Kappa versus vehicle) on bleeding time were assessed by comparing absolute bleeding time (left panels) and percent change in bleeding time (right panels) versus vehicle. as Treatments #1 and 2 in Study Session #1, and vehicle-αFXI-18623p IgG4 HC (S228P) (E1)/LC Kappa as Treatments #1 and 2 in Study Session #2, using unilateral paired Student's t-test.

Fig. 17A shows concentration-time profiles following IV administration of aFXI-18623p IgG4 HC (S228P) (E1)/LC kappa to rhesus monkeys. Plasma concentration-time profiles for aFXI-18623p IgG4 HC (S228P)(E1)/LC kappa in rhesus monkeys are presented. There were four animals per group for each dose. Each line represents the average for a specific group.

Fig. 17B shows APPT time profiles in rhesus monkeys, APPT time profiles for aFXI-18623p IgG4 HC (S228P) (E1)/LC kappa are presented for each dose group. There were four animals per group for each dose. Each symbol represents an individual APPV time profile of the animal at each time point. Each line represents the average for a specific group.

Detailed Description of the Invention

The present invention relates to anti-coagulation factor XI antibodies that bind to the apple 3 domain of coagulation factor XI (FXI). These anti-FXI antibodies are inhibitors of FXI activation by factor XIIa and are suitable for inhibiting blood coagulation and associated thrombosis without impairing hemostasis (antithrombotic indication). For example, anti-FXI antibodies may be used for the treatment and prevention of venous thromboembolism (VTE), stroke prevention in atrial fibrillation (SPAF), or the treatment and prevention of certain thromboembolic disorders associated with medical devices (eg, stents, intravascular stent grafts, catheters (cardiac or venous) ), continuous flow ventricular assist systems (CF-LVADS), hemodialysis, heart-lung machine and extracorporeal membrane oxygenation (ECMO), ventricular assist systems (VADS)). Thus, the anti-FXI antibodies described herein are suitable for therapy for the treatment of a thromboembolic disorder or disease in a patient or individual who requires such therapy.

FXI is a homodimeric serine protease that has the domain structure shown in FIG. 1B and is an integral component of the intrinsic pathway of the coagulation cascade. Zymogen FXI can be cleaved by factor XIIa to its activated form FXIa. FXIa then activates factor IX and ultimately triggers thrombin production and clot formation. The anti-FXI antibodies described herein inhibit the conversion of FXI to FXIa (See FIG. 1A).

Anti-FXI antibody molecules were generated from a fully human synthetic IgG1/kappa library surface-mounted on engineered yeast strains. The library was screened using FXI or FXIa to identify antibodies capable of binding to human FXI with subnanomolar affinity to human FXI and non-human primate (NHP) FXI and not binding to human and NHP plasma kallikrein (a protein showing 56% amino acid identity with FXI), or with other human coagulation cascade proteins (FII//IIa, FVII/VIIa, FIX/IXa, FX/Xa and FXII/XIIa). Two antibodies with these properties have been identified: aFXI-18611p and aFXI-18623p. These antibodies were fully human antibodies containing human kappa (k) light chain and human IgG1 (γ1) isotype heavy chain. The antibodies selectively bind to the FXI zymogen epitope comprising SEQ ID NO:82 and 83 located in the apple 3 domain of FXI. These antibodies also bind to FXIa with an affinity comparable to that of the FXI zymogen.

Antibodies from the aFXI-18611p family contain heavy chain (HC) complementarity determining regions (CDRs) 1, 2, and 3 with the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively , and light chain (LC) CDRs I, 2, and 3 with the amino acid sequences shown in SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively. The aFXI-18611p family includes antibodies containing a heavy chain (HC) variable domain containing the amino acid sequence shown in SEQ ID NO: 21 or 22, and a light chain (LC) variable domain containing the amino acid sequence of SEQ ID NO: 25.

Antibodies from the aFXI-18611 family contain heavy chain (HC) complementarity determining regions (CDRs) 1, 2, and 3 with the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 4, respectively. and light chain (LC) CDRs 1, 2 and 3 with the amino acid sequences shown in SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, respectively. The aFXI-18611 family includes antibodies containing a heavy chain (HC) variable domain containing the amino acid sequence shown in SEQ ID NO: 23 or 24, and a light chain (LC) variable domain containing the amino acid sequence of SEQ ID NO: 25.

Antibodies from the aFXI-18623p family contain CDRs 1, 2, and 3 HC with the amino acid sequences shown in SEQ ID NO: 8, SeQ ID NO: 9 and SEQ ID NO:10, respectively, and CDR 1, 2 and 3 LC with the amino acid sequences the sequences shown in SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, respectively. The aFXI-13716p family includes antibodies containing a heavy chain (HC) variable domain containing the amino acid sequence shown in SEQ ID NO: 28 or 29, and a light chain (LC) variable domain containing the amino acid sequence of SEQ

- 21 043303

ID NO: 30. Antibodies of this family were obtained from a different lineage than the previous families.

The present invention further provides anti-FXI antibodies comprising at least six CDRs from an anti-FXI antibody of the aFXI-18611p family, the aFXI-18611 family or the aFXI18623p family, or embodiments thereof, wherein one or more of the six CDRs have one, two or three amino acid substitutions, insertions, deletions, or combinations thereof, and to methods of using antibodies to treat antithrombotic indications, such as SPAF.

In specific aspects, the anti-FXI antibodies comprise at least an anti-FXI antibody HC variable domain from the aFXI-18611p family, the aFXI-18611 family, or the aFXI-18623p family, or a variant thereof, wherein the HC variable domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, insertions, deletions, or combinations thereof.

In specific aspects, the anti-FXI antibodies comprise at least an anti-FXI antibody LC variable domain from the aFXI-18611p family, the aFXI-18611 family, or the aFXI-18623p family, or a variant thereof, wherein the LC variable domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, insertions, deletions, or combinations thereof.

In specific aspects, the anti-FXI antibodies comprise at least an anti-FXI antibody HC variable domain from the aFXI-18611p family, the aFXI-18611 family, or the aFXI-18623p family, or a variant thereof, wherein the HC variable domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, insertions, deletions, or combinations thereof, and the LC variable domain of an anti-FXI antibody of the aFXI-18611p family, the aFXI-18611 family, or the aFXI-18623p family, or a variant thereof, wherein the variable domain LC contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, insertions, deletions, or combinations thereof.

In specific embodiments, the antibodies herein comprise at least six CDRs from an anti-FXI antibody of the aFXI-18611p family, the aFXI-18611 family, or the aFXI-18623p family, or embodiments thereof, wherein one or more of the six CDRs have one , two or three amino acid substitutions, insertions, deletions, or combinations thereof, and further comprise a heavy chain (HC), which is of the human IgG1, IgG2, IgG3, or IgG4 isotype, and a light chain (LC), which may be of the kappa or human IgG4 isotype. lambda. In other embodiments, the antibodies herein comprise at least six CDRs from an anti-FXI antibody of the aFXI-18611p family, the aFXI-18611 family, or the aFXI-18623p family, or embodiments thereof, wherein one or more of the six CDRs have one, two or three amino acid substitutions, insertions, deletions, or combinations thereof, and may additionally be classified as IgM, IgD, IgA, or IgE. In specific embodiments, a human IgG1, IgG2, IgG3, or IgG4 isotype may include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, insertions, deletions, or combinations thereof.

In specific embodiments, the antibodies may comprise at least six CDRs from an anti-FXI antibody of the aFXI-18611p family, the aFXI-18611 family, or the aFXI-18623p family, or embodiments thereof, wherein one or more of the six CDRs have one, two, or three substitution, insertion, deletion of amino acids or combinations thereof, and additionally contain a constant HC domain, which belongs to the IgG4 isotype. The IgG4 framework provides the antibody with little or no effector function. In a further aspect of the invention, the antibodies may comprise at least six CDRs from an anti-FXI antibody of the aFXI-18611p family, the aFXI-18611 family, or the aFXI-18623p family, or embodiments thereof, wherein one or more of the six CDRs have one, two, or three substitutions, insertions, deletions of amino acids or combinations thereof, and additionally contain a constant HC domain, which belongs to the IgG4 isotype, fused with a variable HC domain, which belongs to the IgG1 isotype. In a further aspect of the invention, the antibodies may comprise at least an HC variable domain and an LC variable domain from an anti-FXI antibody of the aFXI-18611p family, the aFXI-18611 family, or the aFXI-18623p family, or variants thereof, wherein the HC and LC variable domains independently contain 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, insertions, deletions of amino acids or combinations thereof, and additionally contain a constant HC domain, which belongs to the IgG4 isotype. In a further aspect of the invention, the antibodies may comprise at least an HC and LC variable domain from an anti-FXI antibody of the aFXI-18611p family, the aFXI-18611 family, or the aFXI-18623p family, or variants thereof, wherein the HC and LC independently comprise 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, insertions, deletions, or combinations thereof, and additionally contain an HC constant domain that is of the IgG4 isotype.

Antibodies of the present invention further include, but are not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), biparatopic antibodies, fully human antibodies, and chimeric antibodies.

Generally, the amino acid sequence of the heavy chain of an antibody, such as IgG1 or IgG4, has a lysine at the C-terminus of the heavy chain constant domain. In some cases, to improve the homogeneity of the antibody product, the antibody may be prepared without the C-terminal lysine. The anti-FXI antibodies of the present invention include embodiments in which the C-terminal lysine is present and embodiments in which the C-terminal lysine is absent. For example, constant domain

- 22 043303

The IgGl HC may have the amino acid sequence shown in SEQ ID NO: 18 or 19, and the IgG4 HC constant domain may have the amino acid sequence shown in SEQ ID

NO:16 or 17.

In certain embodiments, the N-terminal amino acid of HC may be a glutamine residue. In certain embodiments, the N-terminal amino acid of HC may be a glutamic acid residue. In particular aspects, the N-terminal amino acid is modified to a glutamic acid residue.

The present invention further provides anti-FXI antigen binding fragments that contain at least six CDRs from an anti-FXI antibody of the αFXI-18611p family, the aFXI-18611 family, or the aFXI-18623p family, or embodiments thereof, wherein one or more of the six CDRs have one, two or three amino acid substitutions, insertions, deletions or combinations thereof.

The present invention further provides anti-FXI Fab fragments that comprise at least six CDRs from an anti-FXI antibody of the aFXI-18611p family, the aFXI-18611 family, or the aFXI-18623p family, or embodiments thereof, wherein one or more of the six CDRs have one, two or three amino acid substitutions, insertions, deletions, or combinations thereof.

The present invention further provides anti-FXI antibodies that contain at least six CDRs from an anti-FXI antibody of the aFXI-18611p family, the aFXI-18611 family, or the aFXI-18623p family, or embodiments thereof, wherein one or more of the six CDRs have one, two or three amino acid substitutions, insertions, deletions or combinations thereof, and to their antigen-binding fragments that contain the Fc region, and to methods of using them.

The present invention further provides anti-FXI Fab' fragments that comprise at least six CDRs from an anti-FXI antibody of the aFXI-18611p family, the aFXI-18611 family, or the aFXI-18623p family, or embodiments thereof, wherein one or more of six CDRs have one, two, or three amino acid substitutions, insertions, deletions, or combinations thereof.

The present invention further provides F(ab') ₂ to FXI that contain at least six CDRs from an anti-FXI antibody of the aFXI-18611p family, the aFXI-18611 family, or the aFXI-18623p family, or embodiments thereof, where one or more of the six CDRs have one, two or three amino acid substitutions, insertions, deletions, or combinations thereof.

The present invention further provides anti-FXI Fv fragments that comprise at least six CDRs from an anti-FXI antibody of the aFXI-18611p family, the aFXI-18611 family, or the aFXI-18623p family, or embodiments thereof, wherein one or more of six CDRs have one, two, or three amino acid substitutions, insertions, deletions, or combinations thereof.

The present invention further provides anti-FXI scFv fragments that contain at least six CDRs from an anti-FXI antibody of the aFXI-18611p family, the aFXI-18611 family, or the aFXI-18623p family, or embodiments thereof, wherein one or more of the six CDRs have one, two, or three amino acid substitutions, insertions, deletions, or combinations thereof.

The present invention further provides anti-FXI domain antibodies that comprise at least three HC CDRs or three LC CDRs from an anti-FXI antibody of the aFXI-18611p family, the aFXI-18611 family, or the aFXI-18623p family, or embodiments thereof, wherein one or more of the HC or LC CDRs have one, two or three amino acid substitutions, insertions, deletions, or combinations thereof. In an embodiment of the invention, the domain antibody is a single domain antibody or nanobody. In an embodiment of the invention, the domain antibody is a nanobody comprising at least a CDR from the aFXI-18611p family, the aFXI-18611 family, or the aFXI18623p family, or embodiments thereof, where one or more of the CDRs have one, two, or three substitutions , insertions, deletions of amino acids or combinations thereof.

The present invention further provides bivalent FXI antibodies that contain at least six CDRs from an anti-FXI antibody of the aFXI-18611p family, the aFXI-18611 family, or the aFXI-18623p family, or embodiments thereof, wherein one or more of the six CDRs have one , two or three amino acid substitutions, insertions, deletions, or combinations thereof.

The present invention further relates to bispecific antibodies and antigen binding fragments having binding specificity for FXI and other antigen of interest, and methods of using them.

Biparatope antibodies are antibodies with binding specificity to different epitopes on the same antigen. The present invention further provides biparatope antibodies with a first heavy/light chain pair from a first antibody that contains at least six CDRs from an anti-FXI antibody of the aFXI-18611p family, the aFXI-18611 family, or the aFXI-18623p family, or embodiments thereof, wherein one or more of the six CDRs have one, two or three amino acid substitutions, insertions, deletions, or combinations thereof, and the second heavy/light chain pair from a second antibody with specificity for an FXI epitope that is different from the epitope recognized by the first heavy/light pair chain.

- 23 043303

The present invention further relates to anti-FXI antibodies and antigen-binding fragments thereof comprising a first heavy/light chain pair from an antibody that contains at least six CDRs from an anti-FXI antibody of the aFXI-18611p family or the aFXI-18611 family or embodiments thereof, wherein one or more of the CDRs having one, two or three amino acid substitutions, insertions, deletions, or combinations thereof, and a second heavy/light chain pair from an antibody that contains at least six CDRs from an aFXI-18623p family antibody or embodiments thereof, wherein one or more of the CDRs have one, two or three amino acid substitutions, insertions, deletions, or combinations thereof.

The present invention further provides anti-FXI diabodies that contain at least six CDRs from an anti-FXI antibody of the aFXI-18611p family, the aFXI-18611 family, or the aFXI-18623p family, or embodiments thereof, wherein one or more of the six CDRs have one , two or three amino acid substitutions, insertions, deletions, or combinations thereof.

An antibody that contains at least six CDRs from an anti-FXI antibody of the aFXI18611p family, the aFXI-18611 family, or the aFXI-18623p family, or embodiments thereof, where one or more of the six CDRs have one, two, or three substitutions, insertions, deletions amino acids or combinations thereof, can be modified in some way such that it retains at least 10% of the FXI binding activity (compared to the parent antibody, i.e., an antibody from the corresponding aFXI-18611p family, aFXI-18611 family or aFXI-18623р), while its activity is expressed in moles. Preferably, the antibody or antigen binding fragment of the invention retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more binding affinity for FXI compared to the parent antibody. It is also contemplated that the antibody or antigen binding fragment of the invention may include conservative or non-conservative amino acid substitutions (referred to as conservative variants or functionally conserved antibody variants) that do not substantially alter its biological activity.

The present invention further provides isolated anti-FXI antibodies that comprise at least six CDRs from an anti-FXI antibody of the aFXI-18611p family, the aFXI-18611 family, or the aFXI-18623p family, or embodiments thereof, wherein one or more of the six CDRs have one, two or three substitutions, insertions, deletions of amino acids or combinations thereof, and to their antigen-binding fragments and methods of using them, as well as to isolated immunoglobulin polypeptide chains and to isolated polynucleotides encoding such polypeptides, and to isolated vectors containing such polynucleotides.

The present invention further provides anti-FXI monoclonal antibodies that contain at least six CDRs from an anti-FXI antibody of the aFXI-18611p family, the aFXI-18611 family, or the aFXI-18623p family, or embodiments thereof, wherein one or more of the six CDRs have one, two or three substitutions, insertions, deletions of amino acids or combinations thereof, and to antigen-binding fragments thereof, as well as monoclonal compositions containing a plurality of isolated monoclonal antibodies.

The present invention further provides chimeric anti-FXI antibodies that contain at least six CDRs from an anti-FXI antibody of the aFXI-18611p family, the aFXI-18611 family, or the aFXI-18623p family, or embodiments thereof, wherein one or more of the six CDRs have one, two or three amino acid substitutions, insertions, deletions or combinations thereof.

The present invention provides fully human anti-FXI antibodies that contain at least six CDRs from an anti-FXI antibody of the aFXI-18611p family, the aFXI-18611 family, or the aFXI-18623p family, or embodiments thereof, wherein one or more of the six CDRs have one, two, or three amino acid substitutions, insertions, deletions, or combinations thereof, both to their antigen-binding moieties and to their methods of use. In an embodiment of the invention, the fully human anti-FXI antibody or antigen-binding fragment thereof is a product isolated from a transgenic animal, e.g., a mouse (e.g., a HUMAB mouse, see, e.g., US Pat. Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016; 5,770,429 ; 5789650; 5814318; 5874299 and 5877397; and Harding, et al., (1995) Ann. NY Acad. Sci. 764:536-546; or XENOMOUSE, see for example, Green et al., 1999, J. Immunol. Methods 231:11-23), which has been genetically modified to produce a complete set of human immunoglobulin genes; or a product isolated from a phage or virus that expresses immunoglobulin chains from a fully human anti-FXI antibody or antigen-binding fragment thereof.

In some embodiments, different constant domains may be added to the V _L and V H regions derived from the CDRs provided herein. For example, if a particular target application of an antibody (or fragment) of the present invention is to cause a change in effector functions, a heavy chain constant domain other than human IgG1 may be used, or an IgG1/IgG4 hybrid may be used.

Although human IgG1 antibodies provide long half-life and effector functions such as complement activation and antibody-dependent cellular cytotoxicity, such properties may not be desirable for all applications of the antibody. In such cases, for example, you can use

- 24 043303 constant domain of human IgG4. The present invention relates to anti-FXI antibodies and antigen-binding fragments thereof that contain an IgG4 constant domain, such as antagonistic human anti-FXI antibodies and fragments, and methods of using them. In one embodiment, the IgG4 constant domain may differ from the native human IgG4 constant domain (Swiss-Prot accession number P01861,l) at a position corresponding to position 228 in the EU system and position 241 in the KABAT system, where the native serine is at position 108 ( Ser108) of the HC constant domain is replaced with proline (Pro) to prevent a potential intrachain disulfide bond between the cysteine at position 106 (Cys106) and the cysteine at position 109 (Cys109), corresponding to positions Cys226 and Cys229 in the EU system and positions Cys239 and Cys242 in the KABAT system), which may prevent proper intrachain disulfide bond formation. See Angal et al. Mol. Imunol. 30:105 (1993); see also (Schuurman et. al., Mol. Immunol. 38: 1-8, (2001); SEQ ID NO: 14 and 41). In other cases, a modified IgG1 constant domain that has been modified to reduce effector function may be used, for example, the IgG1 isotype may include substitutions with IgG2 residues at positions 233-236 and IgG4 residues at positions 327, 330, and 331 to further reduce ADCC and CDC (Armour et al., Eur J Immunol. 29 (8): 2613-24 (1999); Shields et al., J Biol Chem. 276 (9): 6591-604 (2001)). In another embodiment, the IgG HC is modified to lack N-glycosylation of the asparagine residue (Asn) at approximately position 297. The consensus sequence for N-glycosylation is Asn-Xaa-Ser/Thr (where Xaa is any amino acid other than Pro); in IgG1, the consensus sequence for Nglycosylation is Asn-Ser-Thr. The modification can be made by replacing the codon for Asn at position 297 in the nucleic acid molecule encoding HC with a codon for another amino acid, for example Gln.

Alternatively, the codon for Ser may be replaced by a codon for Pro, or the codon for Thr may be replaced by any codon except the codon for Ser. Such modified IgG1 molecules have little or no effector function. Alternatively, all three codons are modified.

In an embodiment of the invention, anti-FXI antibodies that contain at least six CDRs from an anti-FXI antibody of the aFXI-18611p family, the aFXI-18611 family, or the aFXI-18623p family, or embodiments thereof, wherein one or more of the six CDRs have one, two or three amino acid substitutions, insertions, deletions, or combinations thereof, have a complete tetrameric structure with two light chains and two heavy chains including constant regions. The variable regions of each light/heavy chain pair form the binding site of the antibody. Thus, as a rule, an intact antibody has two binding sites. With the exception of bispecific antibodies, the two binding sites are usually the same.

In specific embodiments, the present invention relates to the anti-FXI antibodies shown in table. 1.

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Table 1

Family yours Antibody Heavy chain (NS) SEQ ID NO: Light Chain (LC) SEQ ID NO: αΕΧΙ- 18611r aFXI-18611p IgG4 NS (S228P)(QI)(M105)/LC kappa 33 26 aFXI-18611p IgG4 NS (S228P)(El)(M105)/LC kappa 35 26 aFXI-18611p IgGl HC (Ql)(M105)/LC kappa 45 26 aFXI-18611p IgGl HC (El) (Ml 05)/LC kappa 47 26 aFXI-18611p IgG4 HC (S228P)(QI)(M105)(K-)/LC kappa 57 26 aFXI-18611p IgG4 HC (S228P)(El)(M105)(K-)/LC kappa 59 26 aFXI-18611p IgGl HC (QI)(M105)(K)/LC kappa 69 26 aFXI-18611p IgGl HC (El)(M105)(K)/LC kappa 71 26 aFXI- 18611 aFXI-18611 IgG4 HC (S228P)(QI)(L105)/LC kappa 37 26 aFXI-18611 IgG4 HC (S228P) (El) (L105)/LC kappa 39 26 aFXI-18611 IgGl HC (Ql) (L105)/LC kappa 49 26 aFXI-18611 IgGl HC (El)(L105)/LC kappa 51 26 aFXI-18611 IgG4 HC (S228P)(QI)(L105)(K-)/LC kappa 61 26 aFXI-18611 IgG4 HC 63 26 (S228P)(El)(L105)(K-)/LC kappa aFXI-18611 IgGl HC (Ql) (L105) (K)/LC kappa 73 26 aFXI-18611 IgGl HC (El) (L105) (K)/LC kappa 75 26 aFXI- 18623rub aFXI-18623p IgG4 HC (S228P)(Ql)/LC kappa 41 31 aFXI-18623p IgG4 HC (S228P) (El)/LC kappa 43 31 aFXI-18623p IgGl HC (Q1)/LC kappa 53 31 aFXI-18623p IgGl HC (E1)/LC kappa 55 31 aFXI-18623p IgGl HC (S228P)(Ql)(K-)/LC kappa 65 31 aFXI-18623p IgG4 HC (S228P)(El)(K-)/LC kappa 67 31 aFXI-18623p IgGl HC (Ql)(K-)/LC kappa 77 31 aFXI-18623p IgGl HC (El)(K-)/LC kappa 79 31

Epitope mapping by hydrogen-deuterium exchange mass spectrometry (HDX-MS), as described in Example 3, showed that anti-FXI antibodies containing the above HC and LC CDRs bind to a specific epitope on the apple 3 domain containing SEQ ID NO: 82 and SEQ ID NO: 83.

Thus, the antibodies described herein bind to the apple 3 domain of FXI

- 26 043303 and inhibit the activation of FXI by FXIIa, and also behave as allosteric competitive inhibitors of FIX activation by FXIa. Epitope mapping results indicate that the aFXI-18623p family recognition region on Apple 3 overlaps with the FIX-binding exosite in

FXIa.

Pharmaceutical compositions and administration

To prepare pharmaceutical or sterile compositions of an anti-FXI antibody or binding fragment thereof, the antibody or antigen binding fragments thereof are mixed with a pharmaceutically acceptable carrier or excipient. See, for example, Remington's Pharmaceutical Sciences and U.S. Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984) and continuously updated online United States Pharmacopoeia (USP) 12601 Twinbrook Parkway, Rockville, MD 208521790, USA.

Therapeutic and diagnostic agent formulations can be prepared by admixture with suitable carriers, excipients or stabilizers in the form of, for example, lyophilized powders, thick suspensions, aqueous solutions or suspensions (see, for example, Hardman, et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams and Wilkins, New York, NY; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY).

In a further embodiment, a composition comprising an antibody or antibody fragment described herein is administered to an individual in accordance with the 2017 Physician's Desk Reference (Thomson Healthcare; 75th edition (November 1, 2002)).

The route of administration may vary. A suitable route of administration is preferably parenteral or subcutaneous. Other routes of administration may include oral, transmucosal, intradermal, direct intraventricular, intravenous, intranasal, inhalation, insufflation or intra-arterial.

In certain embodiments, the anti-FXI antibody or antigen-binding fragment thereof can be administered by an invasive route, such as injection. In additional embodiments of the invention, an anti-FXI antibody or antigen-binding fragment thereof, or a pharmaceutical composition thereof, can be administered intravenously, subcutaneously, intra-arterially, or by inhalation, using an aerosol. Administration by non-invasive routes (eg, orally; eg, in a pill, capsule, or tablet) is also within the scope of the present invention.

The compositions can be administered using medical devices known in the art. For example, the pharmaceutical composition of the invention can be administered by injection using a hypodermic needle, for example, using a prefilled syringe or auto-injector.

The pharmaceutical compositions described herein can also be administered using a needle-free hypodermic injection device, such as the devices described in US Pat. Nos. 6,620,135; 6096002; 5399163; 5383851; 5312335; 5064413; 4941880; 4790824 or 4596556.

The pharmaceutical compositions described herein can also be administered by infusion. Examples of well-known implants and modules for administering pharmaceutical compositions include US Pat. No. 4,487,603, which describes an implantable microinfusion pump for controlled rate drug dispensing; US Patent No. 4,447,233, which describes a drug infusion pump for delivering drugs at a precise infusion rate; US Pat. No. 4,447,224, which describes an implantable variable-flow infusion device for continuous drug delivery; US Patent No. 4,439,196, which describes an osmotic drug delivery system with multi-chamber compartments. Many other such implants, delivery systems and modules are well known to those skilled in the art.

The schedule of administration depends on several factors, including the rate of circulation of the therapeutic antibody in the serum or tissue, the extent of symptoms, the immunogenicity of the therapeutic antibody, and the availability of target cells in the biological matrix. Preferably, the dosage regimen delivers sufficient amounts of the therapeutic antibody to effectively improve the target disease state while minimizing unwanted side effects. Thus, the amount of biological agent delivered depends in part on the specific therapeutic antibody and the severity of the condition being treated. Guidelines for selecting appropriate doses of therapeutic antibodies are available (see, for example, Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, NY; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY; Baert et al (2003) New Engl J Med 348:601-608; Milgrom et al (1999) New Engl J Med 341:1966-1973 Slamon et al (2001) New Engl J Med 344:783-792 Beniaminovitz et al (2000) New Engl J Med 341:1966-1973 Med 342:613-619 Ghosh et al (2003) New Engl J Med 348:24-32 Lipsky et al (2000) New Engl J Med 342:613-619

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Med. 343:1594-1602).

Dosage regimens are adjusted to provide the optimal desired response (eg, therapeutic response). For example, it may be administered in a single dose, several divided doses may be administered over time, or the dose may be proportionally decreased or increased as indicated by the need of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in unit dosage form for ease of administration and dose uniformity. Unit dosage form, as used herein, refers to physically discrete units suitable as unitary dosages for individuals to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect, in combination with the required pharmaceutical carrier. The specification for the unit dosage forms described herein is dictated by and is directly dependent on (a) the unique characteristics of the antibody or antibody binding fragment and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the field of coupling such active molecules for treatment sensitivity in individuals (see, for example, Yang, et al. (2003) New Engl. J. Med. 349:427-434; Herold, et al. (2002) New Engl. J. Med. 346:16921698; Liu , et al (1999) J Neurol Neurosurg Psych 67:451-456; Portielji, et al (20003) Cancer Immunol Immunother 52:133-144).

Sets

Further provided are kits containing one or more components, which include, by way of non-limiting examples, an anti-FXI antibody or antigen-binding fragment described herein, in association with one or more additional components, including, by way of non-limiting examples, an additional therapeutic agent described in this document. The antibody or fragment and/or therapeutic agent can be formulated as a pure composition or in combination with a pharmaceutically acceptable carrier in a pharmaceutical composition.

In one embodiment, the kit includes an anti-FXI antibody or antigen binding fragment thereof or a pharmaceutical composition thereof in one container (eg, a sterile glass or plastic vial) and an additional therapeutic agent in another container (eg, a sterile glass or plastic bottle).

In another embodiment, the kit contains a combination of the invention comprising an anti-FXI antibody or antigen-binding fragment thereof or a pharmaceutical composition thereof in combination with one or more therapeutic agents formulated together, optionally in a pharmaceutical composition, in one common container.

If the kit includes a pharmaceutical composition for parenteral administration to an individual, the kit may include a device for performing such administration. For example, the kit may include one or more hypodermic needles or other injection devices as described above. Thus, the present invention relates to a kit containing an injection device and an anti-FXI antibody or antigen-binding fragment thereof, for example, where the injection device contains the antibody or fragment or where the antibody or fragment is contained in a separate reservoir.

The kit may include a package insert, including information regarding the pharmaceutical compositions and dosage forms in the kit. Generally, such information assists patients and physicians in using the enclosed pharmaceutical compositions and dosage forms effectively and safely. For example, the package insert may provide the following information regarding the combination of the invention: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and use, contraindications, warnings, precautions, adverse reactions, overdose, proper dosage and administration, dosage form, suitable conditions storage, links, manufacturer/distributor information and patent information.

Methods for producing antibodies and their antigen-binding fragments

Antibodies to FXI and fragments thereof described herein can also be produced recombinantly. In this embodiment, nucleic acids encoding antibody molecules can be inserted into a vector (plasmid or viral) and transfected or transformed into a host cell, in which they can be expressed and secreted from the host cell. There are several methods known in the art by which recombinant antibodies can be produced.

Mammalian cell lines available as hosts for the expression of antibodies or fragments described herein are well known in the art and include many immortalized cell lines available in the American Type Culture Collection (ATCC). These lines include, but are not limited to, Chinese hamster ovary (CHO) cells, NSO cells, SP2 cells, HeLa cells, newborn hamster kidney (BHK) cells, monkey kidney (COS) cells, human hepatocellular carcinoma cells (eg, Hep G2), A549 cells, 3T3 cells, human embryonic kidney 293 (HEK-293) cells and a number of other cell lines. Particularly preferred adhesives

- 28 043303 precise lines are selected by identifying cell lines with high expression levels. Other cell lines that can be used are insect cell lines such as Sf9 cells, amphibian cells, bacterial cells, plant cells, filamentous fungal cells (eg Trichoderma reesei), and yeast cells (eg Saccharomyces cerevisiae or Pichia pastoris) . In particular aspects, the host cell may be a prokaryotic host cell, such as E. coli.

When recombinant expression vectors containing a nucleic acid molecule encoding a heavy chain or antigen-binding portion or a fragment thereof, a light chain and/or an antigen-binding fragment thereof are introduced into host cells, antibodies are produced by culturing the host cells under conditions and for a period of time, sufficient to express the antibody in the host cells or, more preferably, secrete the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium and further purified or processed to produce the antibodies of the invention.

In specific aspects, host cells are transfected with an expression vector containing a nucleic acid molecule encoding an HC and LC comprising at least the CDRs of the HC and LC from an anti-FXI antibody aFXI-18611p family, aFXI-18611 family or aFXI-18623p family, or embodiments thereof, wherein one or more of the six CDRs have one, two or three amino acid substitutions, insertions, deletions, or combinations thereof, and/or wherein the HC and/or LC variable region backbone comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, insertions, deletions, or combinations thereof.

In specific aspects, host cells are transfected with a first expression vector containing a nucleic acid molecule encoding an HC comprising at least a CDR of an HC from an anti-FXI antibody aFXI-18611p family, aFXI-18611 family or aFXI-18623p family, or embodiments thereof where one or more of the six CDRs have one, two or three amino acid substitutions, insertions, deletions or combinations thereof, and/or where the HC and/or LC variable region backbone contains 0, 1, 2, 3, 4, 5, 6 , 7, 8, 9 or 10 amino acid substitutions, insertions, deletions, or combinations thereof, and a second expression vector containing a nucleic acid molecule encoding an LC containing at least a CDR LC from an anti-FXI family antibody aFXI-18611p, aFXI family -18611 or the aFXI-18623p family, or embodiments thereof, wherein one or more of the six CDRs have one, two or three amino acid substitutions, insertions, deletions, or combinations thereof, and/or where the HC and/or LC variable region framework contains 0 , 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, insertions, deletions, or combinations thereof.

In specific embodiments, the HC and LC are expressed as a fusion protein in which the N-terminus of the HC and LC is fused to a leader sequence to facilitate transport of the antibody through the secretory pathway. Examples of leader sequences that may be used include MSVPTQVLGLLLLWLTDARC (SEQ ID NO:14) or MEWSWVFLFFLSVTTGVHS (SEQ ID NO:15).

The NS of the exemplary antibodies herein can be encoded by a nucleic acid molecule with the nucleotide sequence shown in SEQ ID NO: 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62 , 64, 66, 68, 70, 72, 74, 76, 78 or 80.

The exemplary antibody LCs herein may be encoded by a nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO:27 or 32.

The present invention further relates to a plasmid or viral vector containing a nucleic acid molecule with the nucleotide sequence SEQ ID NO: 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62 , 64, 66, 68, 70, 72, 74, 76, 78 or 80. The present invention further provides a plasmid or viral vector containing a nucleic acid molecule encoding an HC from an anti-FXI antibody of the aFXI-18611p family, the aFXI-18611 family, or aFXI18623p family, or embodiments thereof, wherein one or more of the six CDRs have one, two or three amino acid substitutions, insertions, deletions, or combinations thereof, and/or wherein the HC and/or LC variable region framework contains 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, insertions, deletions, or combinations thereof, and a nucleic acid molecule encoding an LC from an anti-FXI antibody of the aFXI-18611p family, the aFXI-18611 family, or the aFXI-18623p family , or embodiments thereof, wherein one or more of the six CDRs have one, two or three amino acid substitutions, insertions, deletions, or combinations thereof, and/or wherein the HC and/or LC variable region framework contains 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, insertions, deletions, or combinations thereof.

The present invention further relates to a plasmid or viral vector containing a nucleic acid molecule encoding an HC from an anti-FXI antibody of the aFXI-18611p family, aFXI-18611 family or aFXI-18623p family, and a plasmid or viral vector containing a nucleic acid molecule encoding an LC from anti-FXI antibodies of the aFXI-18611p family, aFXI18611 family, or aFXI-18623p family.

The present invention further relates to a host cell containing one or more plasmids or viral vectors containing a nucleic acid molecule encoding an HC from an anti-FXI antibody aFXI-18611p family, aFXI-18611 family or aFXI-18623p family, or embodiments thereof, wherein one or more of the six CDRs have one, two, or three substitutions, insertions, or deletions

- 29 043303 amino acids or combinations thereof, and/or where the framework of the variable region HC and/or LC contains 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, insertions, deletions of amino acids or their combinations, and a nucleic acid molecule encoding an LC from an anti-FXI antibody of the aFXI-18611p family, the aFXI-18611 family, or the aFXI-18623p family, or embodiments thereof, wherein one or more of the six CDRs have one, two, or three substitutions, insertions, amino acid deletions or combinations thereof, and/or wherein the HC and/or LC variable region framework contains 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, insertions, deletions, or combinations thereof. In specific embodiments, the host cell is a CHO or HEK-293 host cell.

Antibodies can be recovered from the culture medium using standard protein purification techniques. Additionally, the expression of antibodies of the invention (or other constituent groups thereof) from production cell lines can be increased using a number of known methods. For example, the glutamine synthetase gene expression system (GS system) is a common approach to increase expression under certain conditions.

Typically, glycoproteins produced in a particular cell line or transgenic animal will have a glycosylation pattern characteristic of glycoproteins produced in the cell line or transgenic animal (See, for example, Croset et al., J. Biotechnol. 161: 336-348 ( 2012) Thus, the specific glycosylation pattern of an antibody will depend on the specific cell line or transgenic animal used to produce the antibody.However, all antibodies encoded by nucleic acid molecules provided herein or containing amino acid sequences provided herein constitute this invention, regardless of the glycosylation pattern that the antibodies may have.

The following examples are intended to facilitate further understanding of the present invention.

General methods

Standard molecular biology techniques are described by Sambrook, Fritsch and Maniatis (1982 & 1989 2nd Edition, 2001 3rd Edition) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sambrook and Russell (2001) Molecular Cloning, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Wu (1993) Recombinant DNA, Vol. 217, Academic Press, San Diego, CA). Standard methods are also described in Ausbel, et al. (2001) Current Protocols in Molecular Biology, Vols.14, John Wiley and Sons, Inc. New York, NY, which covers bacterial cloning and DNA mutagenesis (Vol. 1), mammalian and yeast cloning (Vol. 2), glycoconjugates and protein expression (Vol. 3), and bioinformatics (Vol. 4).

Methods for protein purification have been described, including immunoprecipitation, chromatography, electrophoresis, centrifugation and crystallization (Coligan, et al. (2000) Current Protocols in Protein Science, Vol. 1, John Wiley and Sons, Inc., New York). Chemical analysis, chemical modification, post-translational modification, production of fusion proteins, protein glycosylation are described (see, for example, Coligan, et al. (2000) Current Protocols in Protein Science, Vol. 2, John Wiley and Sons, Inc., New York ; Ausubel, et al. (2001) Current Protocols in Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY, NY, pp. 16.0.516,22,17; Sigma-Aldrich, Co. (2001) Products for Life Science Research, St. Louis, MO; pp. 45-89; Amersham Pharmacia Biotech (2001) BioDirectory, Piscataway, N.J., pp. 384-391). The preparation, purification and fragmentation of polyclonal and monoclonal antibodies are described (Coligan, et al. (2001) Current Proceedings in Immunology, Vol. 1, John Wiley and Sons, Inc., New York; Harlow and Lane (1999) Using Antibodies, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane, supra). Standard methods are available for characterizing ligand/receptor interactions (see, for example, Coligan, et al. (2001) Current Protocols in Immunology, Vol. 4, John Wiley, Inc., New York).

Monoclonal, polyclonal and humanized antibodies can be produced (see, for example, Sheperd and Dean (eds.) (2000) Monoclonal Antibodies, Oxford Univ. Press, New York, NY; Kontermann and Dubel (eds.) (2001) Antibody Engineering, Springer-Verlag, New York; Harlow and Lane (1988) Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 139-243; Carpenter, et al. (2000) J. Immunol. 165: 6205; He, et al. (1998) J. Immunol. 160:1029; Tang et al. (1999) J. Biol. Chem. 274: 27371-27378; Baca et al. (1997) J. Biol. Chem. 272:10678-10684; Chothia et al (1989) Nature 342:877883; Foote and Winter (1992) J Mol Biol 224:487-499; US Patent No. 6329511).

An alternative to humanization is the use of human antibody libraries located on phages or human antibody libraries in transgenic mice (Vaughan et al. (1996) Nature Biotechnol. 14:309-314; Barbas (1995) Nature Medicine 1:837-839; Mendez et al. al (1997) Nature Genetics 15:146–156; Hoogenboom and Chames (2000) Immunol. Today 21:371–377; Barbas et al (2001) Phage Display: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Kay et al. (1996) Phage Display of Peptides and Proteins: A Laboratory Manual, Academic Press, San Diego, CA; de Bruin et al. (1999) Nature Biotechnol. 17:397-399).

Antibodies can be conjugated, for example, with small drug molecules, enzymes, liposomes, polyethylene glycol (PEG). Antibodies are suitable for therapeutic, diagnostic kits or other purposes and include antibodies coupled, for example, to dyes, radioactive isotopes, enzymes or metals, for example colloidal gold (see, for example, Le Doussal et al.

(1991) J. Immunol. 146:169-175; Gibellini et al. (1998) J. Immunol. 160:3891-3898; Hsing and Bishop (1999)J.

Immunol. 162:2804-2811; Everts et al. (2002) J. Immunol. 168:883-889).

Flow cytometry techniques are known, including fluorescence-activated cell sorting (FACS) (see, for example, Owens, et al. (1994) Flow Cytometry Principles for Clinical Laboratory Practice, John Wiley and Sons, Hoboken, NJ; Givan (2001) ) Flow Cytometry, 2nd ed.; Wiley-Liss, Hoboken, NJ; Shapiro (2003) Practical Flow Cytometry, John Wiley and Sons, Hoboken, NJ). Fluorescent reagents suitable for modifying nucleic acids are available, including primers and probes for nucleic acids, polypeptides, and antibodies, for use, for example, as diagnostic reagents (Molecular Probes catalog (2003), Molecular Probes, Inc., Eugene, OR ; catalog Sigma-Aldrich (2003), St. Louis, MO).

Standard techniques for the histology of the immune system have been described (see, for example, Muller-Harmelink (ed.) (1986) Human Thymus: Histopathology and Pathology, Springer Verlag, New York, NY; Hiatt, et al. (2000) Color Atlas of Histology, Lippincott, Williams, and Wilkins, Phila, PA; Louis, et al. (2002) Basic Histology: Text and Atlas, McGraw-Hill, New York, NY).

Software packages and databases are available to determine, for example, antigenic fragments, leader sequences, protein folding, functional domains, glycosylation sites and for sequence alignments (see, for example, GenBank, Vector NTI® Suite (Informax, Inc, Bethesda, MD ); GCG Wisconsin Package (Accelrys, Inc., San Diego, CA); DeCypher® (TimeLogic Corp., Crystal Bay, Nevada); Menne, et al. (2000) Bioinformatics 16: 741-742; Menne et al. ( 2000) Bioinformatics Applications Note 16:741-742 Wren et al (2002) Comput Methods Programs Biomed 68:177-181 von Heijne (1983) Eur J Biochem 133:17-21 von Heijne ( 1986) Nucleic Acids Res 14:4683-4690).

Human zymogen FXI and FIX can be purchased from Haematologic Technologies, Inc. Essex Junction, VT; high molecular weight (HMW) kininogen can be purchased from Enzyme Research Laboratories, South Bend, IN, and ellagic acid can be purchased from Pacific Hemostasis, ThermoFisher, Waltham, MA.

Example 1.

In this example, the binding kinetics of anti-FXI antibodies aFXI-18611 IgG4 HC (S228P)(E1) (L105)/LC Kappa and aFXI-18623p IgG4 HC (S228P)(Q1)/LC Kappa and either human FXI zymogen or FXI zymogen were measured non-human primate (NHP) using the following assays.

Human FXI/FXIa Binding Kinetics Assay Protocol

Binding kinetics and protein-protein interaction affinity between anti-FXI antibodies and human FXI or FXIa zymogen were determined using ProteOn XPR36 (Bio-Rad), an SPR (surface plasmon resonance) based optical biosensor as follows.

The low-density GLC sensor chip was washed through all vertical and horizontal flow channels with 0.5% sodium dodecyl sulfate, 50 mM sodium hydroxide, and 100 mM hydrochloric acid for 60 s at a flow rate of 30 μL/s. The alginate chip surface for all six vertical flow channels (L1-L6) was then activated with lxEDC/sNHS at a flow rate of 30 μL/s for 150 s. A mouse polyclonal antibody targeting human IgG Fc (capture antibody), diluted to 1.25 μg/ml in 10 mM sodium acetate, pH 5.0, was then injected along all six vertical flow channels for 300 s at a flow rate of 25 μl/mL. s to bind approximately 300 response units (RU) of the capture antibody to the activated chip surface onto the flow channel by amine bonding to endogenous lysine. 1 M ethanolamine HCl was then injected along all six vertical flow channels to neutralize remaining reactive surface amines. Anti-FXI antibodies were then injected at 25 μl/min for 60 s, each into a separate vertical flow channel coated with capture antibody (L2, L3, L4, L5, or L6) at a concentration of 5 μg/ml in 10 mM sodium acetate. pH 5.0 to achieve saturation capture levels of approximately 80 PE; 10 mM sodium acetate, pH 5.0 (buffer only), was injected into the vertical flow channel L1 as a reference control.

After capture of anti-FXI antibodies, running buffer (1x HBS-N, 5 mM CaCl ₂ , 0.005% P20, pH 7.4) was injected along all horizontal flow channels (A1-A6) for 5 min and allowed to dissociate for 20 min at 25 μL/min to remove any unbound anti-FXI antibodies from the chip surface. To measure the rate of forward reaction (k _a ) of human FXI or FXa with bound anti-FXI antibodies, 6 titers of human FXI or FXIa (0, 0.25, 0.5, 1.0, 2.0, 4.0 nM) were then injected , diluted in run buffer) horizontally into all six vertical flow channels for 8 min; the bound zymogen was then allowed to dissociate for 60 min in running buffer at 25 μl/min to measure dissociation (kd). Binding kinetics and affinity (KD) were determined using dedicated instrument software (Bio-Rad) and are shown in Table. 2.

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Protocol for analysis of FXI/FXIa zymogen binding kinetics in a non-human primate

Binding kinetics and protein-protein interaction affinity between anti-FXI antibodies and FXI or FXIa zymogen in a non-human primate (NHP: cynomolgus and rhesus macaques) were determined using ProteOn XPR36 (Bio-Rad), an SPR (surface plasmonic) optical biosensor resonance).

The low-density GLC sensor chip was washed through all vertical and horizontal flow channels with 0.5% sodium dodecyl sulfate, 50 mM sodium hydroxide, and 100 mM hydrochloric acid for 60 s at a flow rate of 30 μL/s. The alginate chip surface for all six vertical flow channels (L1-L6) was then activated with 1xEDC/sNHS at a flow rate of 30 μL/sec for 150 s. A mouse polyclonal antibody targeting human IgG Fc (capture antibody) diluted to 30 μg/ml in 10 mM sodium acetate, pH 5.0, was then injected along all six vertical flow channels for 150 s at a flow rate of 25 μl/s. to bind approximately 4500 response units (RU) of capture antibody to the activated chip surface onto the flow channel via amine coupling to endogenous lysine.

1 M ethanolamine HCl was then injected along all six vertical flow channels to neutralize remaining reactive surface amines. Anti-FXI antibodies were then injected at 25 μl/min for 60 s, each into a separate vertical flow channel coated with capture antibody (L2, L3, L4, L5, or L6) at a concentration of 0.415 μg/ml in running buffer (1xHBS -N, 5 mM CaCl ₂ , 0.005% P20, pH 7.4), to achieve saturation uptake levels of approximately 40 PE; Running buffer alone was injected into the vertical flow channel L1 as a reference control. After capture of anti-FXI antibodies, running buffer (1 x HBS-N, 5 mM CaCl ₂ , 0.005% P20, pH 7.4) was injected along all horizontal flow channels (A1-A6) for 5 min and allowed to dissociate for 20 min at 25 µl/min to remove any unbound anti-FXI antibodies from the chip surface. To measure the rate of forward reaction (k _a ) of NHP FXI or FXa with bound anti-FXI antibodies, 6 titers of NHP FXI or FXIa (0, 0.25, 0.5, 1.0, 2.0, 4.0 nM) were then injected , diluted in run buffer) horizontally into all six vertical flow channels for 8 min; the bound zymogen FXI or FXIa was then allowed to dissociate for 60 min in running buffer at 25 μl/min to measure dissociation (kd). Binding kinetics and affinity (KD) were determined using dedicated instrument software (Bio-Rad). The results are shown in table. 2.

Table 2. Binding of mAbs aFXI-18623P and aFXI-18611 to FXI/XIa

FXI FXIa Average affinity Average affinity Target K _D + SD (pm ) K _D + SD (pM) aFXI- aFXI aFXI aFXI 18611 -18623r -18611 -18623Р Human 100+38 22.6+2.2 55.4+12.2 37.4+10.4 Javanese 180+70 13.0+5.7 89.2+10.4 19.5+0.6 macaques Macaque- 52.9±9.6 72.2+31.7 175+62.6 149+3.8 rhesus

aFXI-18611=HC (S228P)(El) (L105) IgG4/LC Kappa aFXI

18611 aFXI-1862Зр=NS (S228P)(QI) IgG4/LC Kappa aFXI-18623p

Example 2: Effect of Anti-FXI Antibodies on Activation of FXI to FXIa by FXIIa in the Presence of High Molecular Weight Kininogen (HMK) and Ellagic Acid

To measure the effects of anti-FXI antibodies aFXI-18611 IgG4 HC (S228P) (E1) (L105)/LC Kappa and aFXI-18623p IgG4 HC (S228P) (Q1)/LC Kappa on FXI zymogen activation, related enzymatic assays can be used that measure FXIa-mediated tripeptide fluorophore proteolysis (GPR-AFC) to determine whether antibodies themselves inhibit FXI activation. For these experiments, antibodies to FXI were preincubated with FXI zymogen for 1 h. Activation of FXI to FXIa was induced by the addition of FXIIa in the presence of HMW kininogen and ellagic acid. The catalytic activity of FXIa on the tripeptide fluorophore sustrate was then measured as an indicator for zymogen activation. The coupled assay was also performed in the absence of HMW kininogen as a control. Antibodies to FXI in eleven titrated doses, starting at a concentration of 1 μM with a series of 3-fold dilutions, were preincubated with human FXI (Haematologic Technologies, Inc., Catalog No. HCXI-0150, final concentration 30 nM) and BM kininogen (Enzyme Research Laboratories , Catalog No. HK, final concentration 280 nM) in 50 mM HEPES, 150 mM

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NaCl, 5 mM CaCl2, 0.1% PEG-8000, pH 7.4 for 2 h at 25°C in a Corning 3575 non-binding microplate. The activation reaction was then initiated by adding Pacific Hemostasis aPTT-XL reagent containing ellagic acid (ThermoFisher Scientific, Cat. No. 100403, stock concentration 100 μM, final concentration 2 μM) and re-diluted coagulation factor XIIa (Enzyme Research Laboratories, Cat. No. HFXIIa, final concentration 50 pM). The reaction was performed at 25°C for 1 h and then quenched by the addition of 1 μM corn trypsin inhibitor (Haematologic Technologies, Inc., Cat. No. CTI-01). Newly activated FXIa enzymatic activity was detected by the rate of cleavage of Z-GPR-AFC substrate (Sigma, Cat. No. C0980-10MG, final concentration 150 μM) by continuously monitoring fluorescence at 400/505 nm for 10 min using a Tecan Infinite plate reader. M200. The percent inhibition for each data point was recalculated from the TFU/minute data and analyzed using a four-parameter log(inhibitor) versus response equation using GraphPad Prism software. The results are shown in table. 3.

Activation of FXI to FXIa by FXIIa in the absence of MV kininogen and ellagic acid

Antibodies to FXI in eleven titrated doses, starting at a concentration of 1 μM with a series of 3-fold dilutions, were preincubated with human FXI (Haematologic Technologies, Inc., Cat. No. HCXI-0150, final concentration 30 nM) in 50 mM HEPES, 150 mM NaCl, 5 mM CaCl ₂ , 0.1% PEG-8000, pH 7.4 for 2 hours at 25°C in a Corning 3575 microplate with a non-binding surface. The activation reaction was then initiated by adding freshly diluted coagulation factor XIIa (Enzyme Research Laboratories, Cat. No. HFXIIa, final concentration 15 nM). The reaction was performed at 25°C for 1 h and then quenched by the addition of 1 μM corn trypsin inhibitor (Haematologic Technologies, Inc., Cat. No. CTI-01). Newly activated FXIa enzymatic activity was detected by the rate of cleavage of Z-GPR-AFC substrate (Sigma, Cat. No. C0980-10MG, final concentration 150 μM) by continuously monitoring fluorescence at 400/505 nm for 10 min using a Tecan Infinite plate reader. M200. The percent inhibition for each data point was recalculated from the TFU/minute data and analyzed using a four-parameter log(uinhibitor) versus response equation using GraphPad Prism software. The results are shown in table. 3.

Table 3. Effects of aFXI-18623p and aFXI-18611 on FXI activation by FXIIa

Antibody N FXIIa Activation + VM Kininogen Inhibition (1C ₅₀ , nM) FXIIa Activation without a VM kininogen inhibition (ICgg, nM) aFXI-18611 3 7.6+3.5 34+20 aFXI-18623p 3 6.0+1.1 14+9.5 aFXI-18611=aFXI-18611 IgG4 HC (S228P)(E1) (L105)/LC Kappa aFXI-18623p=aFXI-18623p IgG4 HC (S228P) (QI)/LC Kappa ^IC 50 are given as average - ^SD ' ^{p= 3}

Together, these mechanistic studies demonstrate that these anti-FXI antibodies functionally neutralize FXI by preventing activation of FXI by FXIIa and inhibiting the catalytic activity of FXIa on the native substrate.

Example 3 Epitope mapping of anti-FXI antibodies by hydrogen-deuterium exchange mass spectrometry

The contact regions of aFXI-18611 IgG4 HC (S228P) (E1) (L105)/LC Kappa and aFXI-18623p-IgG4 (S228P) (Q1)/LC Kappa with human FXI were determined using hydrogen-deuterium exchange (HDX) mass spectrometry analysis -MS). HDX-MS measures the incorporation of deuterium into the amide backbone of a protein, and changes in its incorporation are influenced by exposure to a hydrogen solvent. Comparisons of deuterium exchange rates between antigen-only and antibody-bound samples were performed to identify regions of antigen that may be in contact with antibody. Human Factor XI has the amino acid sequence shown in SEQ ID NO:81. Dimeric factor XI was preincubated with antibodies before incubation in deuterium buffer. Deuterium incorporation into factor XI was measured by mass spectrometry.

The regions of human factor XI protected from deuteration by antibodies were DIFPNTVF Epitope-A (Factor XI Residues 185-192; SEQ ID NO:82) and PSTRIKKSKALSG Epitope-B (Factor XI Residues 247-259; SEQ ID NO:83). Fig. 3A and 3B show a heat map of the differences in deuterium labeling of FXI amino acid residues bound to aFXI-18611 IgG4 HC (S228P) (E1) (L105)/LC kappa and aFXI-18623p IgG4 HC (S228P)(Q1)/LC kappa antibodies, respectively. These amino acid sequences are located in the Apple 3 domain of factor XI (Fig. 2). No significant changes in deuteration were observed in the Apple 1, 2, 4 or catalytic domains, indicating that they are not involved in binding to aFXI-18623. Thus, the epitope that is recognized by aFXI18623p-IgG4 (S228P)/kappa contains Epitope A and Epitope B.

Example 4.

FIX is an endogenous protein substrate for FXIa, the active protease of the FXI zymogen. FXIa activates FIX before FIXa, again starting the coagulation cascade. Inhibition of FXIa-mediated FIX activation is one of the potential mechanisms of action (MOAs) for FXI mAbs. To investigate this MOA in detail, enzymatic assays with FXIa were developed using the full-length zymogen FIX.

FXIa protease activity on a small tripeptide substrate

Antibodies to FXI were preincubated with human FXIa (Sekisui Diagnostics, Exton, PA, Cat. No. 4011A, final concentration 100 pM) in 50 mM HEPES, 150 mM NaCl, 5 mM CaCl ₂ , 0.1% PEG-8000, pH 7, 4 for 2 hours at 25°C in a Corning 3575 microplate with a non-binding surface. FXIa enzymatic activity was determined by measuring the rate of cleavage of Z-GPR-AFC substrate (Sigma, Cat. No. C0980-10MG, final concentration 100 μM) by continuously monitoring fluorescence at 400/505 nm for 10 min using a Tecan Infinite plate reader. M200. The final concentrations of eleven titration doses of the antibody started at 1 μM with a series of three-fold dilutions. The percent inhibition for each data point was recalculated from the TFU/minute data and analyzed using a four-parameter log(inhibitor) versus response equation using GraphPad Prism software. The results are shown in table. 4.

Activation of FIX to FIXa via FXIa

FIX is an endogenous protein substrate for FXIa, the active protease of the FXI zymogen. FXIa activates FIX before FIXa, again starting the coagulation cascade. Inhibition of FXIa-mediated FIX activation is one of the potential mechanisms of action (MOAs) for FXI mAbs. To investigate this MOA in detail, enzymatic assays with FXIa were developed using the full-length zymogen FIX.

Antibodies to FXI in eleven titrated doses, starting at a concentration of 1 μM with a series of 3-fold dilutions, were preincubated with human FXIa (Sekisui Diagnostics, Catalog No. 4011A, final concentration 100 pM) in 50 mM HEPES, 150 mM NaCl, 5 mM CaCl ₂ , 0.1% PEG8000, pH 7.4 for 2 hours at 25°C in a Corning 3575 microplate with a non-binding surface. The activation reaction was then initiated by the addition of FIX (Haematologic Technologies, Inc., Catalog No. HCIX-0040-C, final concentration 300 nM) and carried out at 25°C for 1 h, then the reaction was quenched by the addition of 100 nM anti-FXI antibody targeting the catalytic region on the FXI light chain (anti-FXI antibody 076D-M007-H04, described in WO 2013167669). Freshly activated FIXa enzymatic activity was detected by the rate of cleavage of the substrate cyclohexyl-GGR-AFC (CPC Scientific, Catalog No. 839493, final concentration 300 μM) by continuously monitoring fluorescence at 400/505 nm for 10 min using a Tecan Infinite M200 plate reader. The percent inhibition for each data point was recalculated from the TFU/minute data and analyzed using a four-parameter log(inhibitor) versus response equation using GraphPad Prism software. The results are shown in table. 4.

Table 4. Effects of aFXI-18623p and aFXI-18611 on the catalytic activity of FXIa

Antibody N FXIa IC ₅₀ nM (tripeptide substrate) FXIa IC ₅₀ nM (native, full-length substrate) aFXI-18611 >1000 1.0+0.3 aFXI-18623p >1000 0.4+0.2 aFXI-18611=aFXI-18611 IgG4 HC (S228P)(E1) (L105)/LC Kappa aFXI-18623p=aFXI-18623p IgG4 HC (S228P) (QI)/LC Kanna 1^-50 are given as average ⁿ⁼ 3

As shown in table. 4, the antibodies did not inhibit FXIa catalytic activity in an enzyme assay using a synthetic tripeptide fluorophore substrate, but both antibodies were potent inhibitors in an assay using the native full-length substrate. These data are consistent with the behavior of the antibodies as allosteric competitive inhibitors of FIX activation by FXIa, as well as the epitope mapping results from Example 3, suggesting that the antibody recognition region on Apple 3 overlaps with the FIX-binding exosite on FXIa.

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Example 5. Autoactivation of FXI to FXIa on dextran sulfate

Antibodies to FXI of the present invention in eleven titrated doses, starting at a concentration of 1 μM with a series of 3-fold dilutions, were preincubated with human FXI (Haematologic Technologies, Inc., Cat. No. HCXI-0150, final concentration 30 nM) in 50 mM HEPES , 150 mM NaCl, 5 mM CaCl2, 0.1% PEG-8000, pH 7.4 for 2 h at 25°C in a Corning 3575 microplate with a non-binding surface. The autoactivation reaction was then initiated by the addition of dextran sulfate (ACROS, Catalog No. 433240250, approximate molecular weight 800 kDa, final concentration 1 nM). The reaction was carried out at 25°C for 1 h and freshly activated FXIa enzymatic activity was detected by the rate of cleavage of the substrate Z-GPR-AFC (Sigma, Cat. No. C0980-10MG, final concentration 150 μM) by continuously monitoring fluorescence at 400/505 nm in for 10 minutes using a Tecan Infinite M200 tablet reader. The percent inhibition for each data point was recalculated from the TFU/minute data and analyzed using a four-parameter log(inhibitor) versus response equation using GraphPad Prism software. The results are shown in table. 5.

Table 5. Effect of aFXI-18623p and aFXI-18611 on F XI autoactivation

Antibody N Autoactivation FXI IC ₅₀ HM aFXI-18611 2 3.3+0.4 aFXI-18623p 2 5.5+4.0 aFXI-18611=aFXI-18611 IgG4 HC (S228P)(E1) (L105)/LC Kappa aFXI-18623p=aFXI-18623p IgG4 HC (S228P) (QI)/LC Kappa ^IC 50 are given as mean ±SD, n= 3

Example 6.

The ability of anti-FXI antibodies to block clotting in vitro was assessed using the activated partial thromboplastin time (aPTT) assay. Activated partial thromboplastin time (aPTT) is a clotting test that measures the activity of the intrinsic and common clotting pathways.

Activated partial thromboplastin time (APTT) analysis

The test is carried out in plasma with sodium citrate. Human plasma was obtained by collecting blood from healthy donors of both sexes in sodium citrate tubes (Sarstedt coagulation 9NC/10 ml). The blood is centrifuged at 1500xg and the plasma is collected. The aPTT is checked for each individual donor and those with aPTT within the normal range (28-40 sec) are selected, aliquoted and stored at -80°C. Plasma from other species was obtained commercially (Innovative Research, Novi, MI). Test samples are prepared by adding inhibitors or vehicle to the plasma. These spiked samples are incubated (60 min, RT) then placed in a coagulation analyzer (STA-R Evolution, Stago Diagnostica, Parsippany, NJ). Basically, the analyzer performs the following steps: activates FXII by adding ellagic acid (Pacific Hemostasis, ThermoFisher Scientific, Waltham, MA), and then measures the time of clot formation after the sample is recalcified. Inhibition of FXI will cause prolongation of APTT clotting time. The results are shown in table. 6. Data are presented as the percentage increase in time compared to vehicle control clotting time, and the concentration that produces 100% (2x) or 50% (1.5x) increase in clotting time is shown. The APTT results are shown in Fig. 6, 7, 8, 9 and 10.

Table 6

Antibody Human Cynomolgus macaque Rhesus monkey 2x (nM) 1.5 (nM) 2x (nM) 1.5 (nM) 2x (nM) 1.5 (nM) aFXI-18623p 24 19 21 15 22 15 aFXI-18611 37 23 218 42 79 22 aFXI-18611=aFXI-18611 IgG4 HC (S228P)(E1) (L105)/LC Kappa aFXI-18623p=aFXI-18623p IgG4 HC (S228P)(QI)/LC Kappa

Example 7: Surface Plasmon Resonance Assay to Assess Off-Target Binding of Anti-FXI Monoclonal Antibodies to Human Coagulation and NHP Proteins

Surface plasmon resonance assay (Biacore T200) was used to determine potential off-target interactions of the FXI mAbs, aFXI-18611 IgG4 HC (S228P) (E1) (L105)/LC Kappa and aFXI-18623p IgG4 HC (S228P) (Q1)/LC Kappa with other coagulation cascade proteins

- 35 043303 studies of humans and NHP (Table 7). Anti-FXI mAb was captured onto a CM5 sensor chip immobilized using an anti-human IgG (Fc) capture kit (GE Healthcare) at approximately 500 pfu to minimize potential background from co-purified Ig in plasma-derived proteins. A negative control antibody, a respiratory syncytial virus (RSV) monoclonal antibody (mAb), was used as a reference and to reduce background binding of plasma-derived proteins. Binding kinetics were measured using an analytical concentration of FXI of 5 nM; all other proteins of the coagulation cascade were used at an analytical concentration of 500 nM. Single concentration injections were performed (n=2) at 30 μl/min, 25°C, HBSEP+, pH 7.4.

Table 7. Recombinant and plasma-derived proteins of the human coagulation cascade and NHP

The consignment Commonly used No./Catalogue Salesman Elnoe Source No. Name Merck, Sharp Recombinant protein & Dohme Kallikrein with His tag us- 0 0AJF Corp., macaque plasma end. Reference Kenilworth rhesus subsequence NJ USA NCBI: ENN26351 65AJE Merck, Sharp & Dohme Corp., Kenilworth, NJ USA Cynomolgus monkey plasma kallikrein Recombinant protein with a His tag at the end. Reference NCBI sequence: XP_005556538.1 97AJY/HPK 1302 Enzyme Research Laboratories Human plasma prekallikrein Highlighted person from plasma 98AJY/HPKa 1303 Enzyme Research Laboratories Human plasma kallikrein Highlighted person from plasma 42AHG/HCP- 0010 Haematologic Technologies Inc. Human Factors II (os-thrombin) Highlighted person from plasma 50AHK/HCVI 1-0030 Haematologic Technologies Inc. Human Factors VII Highlighted person from plasma 51AH/HCVII A-0031 Haematologic Technologies Inc. Human factor VI1a/Protease Highlighted person from plasma 38AHG/HCIX -0040 Haematologic Technologies Inc. Human Factors IX Highlighted person from plasma 14AJZ/HFIX Enzyme Human Highlighted from plasma

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a 1080 Research Laboratories factor 1Xa/Protease person 15AJZ/HFX1 010 Enzyme Research Laboratories Human Factors X Isolated from plasma person 18AJZ/HFXa 1011 Enzyme Research Laboratories Human Factor Xa/Protease Isolated from plasma person 19AJZ/HFXI I 1212 Enzyme Research Laboratories Human Factors XII Isolated from plasma person 20AJZ/HFXI I 1212a Enzyme Research Laboratories Human Factor X11a/Protease Isolated from plasma person 23AIR/HCXI -0150-C Haematologic Technologies Inc. Human FXI Isolated from plasma person 41AHG/HCP- 0010 Haematologic Technologies Inc. Human factor II (prothrombin) Isolated from plasma person 82AJK/2460 -SE R&D Human FXI with His tag Recombinant protein with a His tag at the end. NSO-derived mouse myeloma cell line. NCBI reference PO3951. 2 3AFE Merck, Sharp & Dohme Corp., Kenilworth, NJ USA mAb IgG4 to RSV SEQ ID NO: 84 (LC) and SEQ ID NO:85 (NS)

Binding kinetics of mAb to factor FXI, aFXI-18611 IgG4 NS (S228P) (E1) (L105)/LC Kappa and aFXI-18623p IgG4 NS (S228P) (Q1)/LC Kappa with FXI of human, cynomolgus and rhesus monkeys and with other human coagulation cascade proteins and NHP were measured as described above and are shown in FIG. 11 and fig. 12). Used Biacore T200 evaluation software to fit the data to a 1:1 binding model to determine the association constant, k _a (M ^-1 sec ^-1 , where M is mole and s is second) and the dissociation constant, kd (s ^-1 ) . These rate constants were used to calculate the equilibrium dissociation constant, KD(M).

aFXI-18611 IgG4 NS (S228P)(E1) (L105)/LC Kappa and aFXI-18623p IgG4 NS (S228P)(Q1)/LC Kappa captured on the chip did not show cross-reaction against non-FXI proteins of the coagulation cascade (Fig. 11 and Fig. 12). These monoclonal antibodies showed the expected levels of strong binding to human, cynomolgus (and rhesus) FXI proteins.

Example 8: Thrombosis model with femoral arteriovenous (AV) shunt in cynomolgus monkey

The antithrombotic efficacy of aFXI-18623p IgG4 HC (S228P) (E1)/LC Kappa antibody was characterized in vivo in the cynomolgus monkey femoral arteriovenous (AV) shunt model developed at the research laboratories of Merck, Sharp & Dohme Corp., Kenilworh, NJ USA and Palo Alto, CA USA.

Study design:

These studies used a repeat design where each animal received 2 shunts over two consecutive test periods (see FIG. 13, Study Design). Monkeys were treated with vehicle without antibody (20 mM sodium acetate, 9% sucrose, pH 5.5) or aFXI18623p IgG4 HC (S228P) (E1)/LC Kappa antibody (dose range 0.01 to 1.0 mg/ kg), during the first and second test periods, respectively. The difference between clot weight measured during the first (vehicle) and second (antibody) test sessions determined antithrombotic efficacy. That is, a greater reduction in clot weight during exposure to aFXI-18623p IgG4 HC (S228P) (E1)/LC Kappa antibody compared to vehicle would indicate a greater antithrombotic effect. Using the repeated paired design described above allows the assessment of antithrombotic efficacy in the animal before and after treatment.

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Details of the AV shunt placement method:

To perform this model, arterial and venous catheters were placed in anesthetized cynomolgus monkeys. These catheters allowed insertion and removal of the AV shunt. AV shunts consisted of a TYGON tube with a piece of silk thread passing through and suspended in the opening of the tube. For AV shunt placement, both the arterial and venous catheters were closed to stop blood flow. The AV shunt was then placed between the two catheters. The timing of catheter placement and removal is indicated in FIG. 13. After the shunt was placed, the catheters were opened and blood flowed through the shunt system, contacting the silk thread. The action of blood coming into contact with the thread promotes the formation of a clot. The AV shunt was left in place for 40 minutes. To remove the AV shunt, both the arterial and venous catheters were closed to stop blood flow through the AV shunt. The shunt was then removed and cut to expose the silk thread and clot. The thrombus was weighed. Data are presented as net weight of the clot, which was determined as the total weight of the clot minus the weight of the silk thread.

Coagulation biomarkers, activated partial thromboplastin time (aPTT) and prothrombin time (PT), as well as circulating levels of plasma aFXI-18623p IgG4 HC (S228P) (E1)/LC Kappa antibody were measured in blood samples collected throughout the experiment, as shown in Fig. 13 APTT and PT were measured in thawed (previously frozen to -80°C) citrated plasma collected from cynomolgus monkeys using a Sta Compact Max coagulation analyzer (Stago Diagnostic, Inc). The Stago analyzer measures clot formation time using an electromagnetic mechanical clot detection system. For APTT analysis, 50 μl of plasma was mixed with 50 μl of ellagic acid mixture (aPTT-XL, Pacific Hemostasis; Fisher Diagnostics catalog no. 10-0402) at 37°C for 3 min. 50 μl of 0.025 M calcium chloride (Sta-CaCl ₂ 0.025 M, Stago Diagnostic, Inc., cat. no. 00367) was added to the mixture and the clot formation time was measured. For RT analysis, 50 μl of plasma was incubated at 37°C for 4 min. The onset of clot formation was initiated by the addition of 100 μl of thromboplastin reagent (Neoplastine Cl Plus 10, Stago Diagnostic, Inc., cat. no. 00667). Plasma was measured as follows. A standard electrochemiluminescence-based hIgG4 immunoassay was used to quantify the antibody in cynomolgus monkey plasma. The assay was performed with biotinylated goat anti-huIgG(H+L) from Bethyl (cat. no. A80-319B) as the capture reagent, and sulfoTAG-tagged mouse anti-human IgG (Fc-specific) from Southern Biotech (cat. no. 9190 -01) as a detection reagent. This assay was qualitative and the lower limit of quantitation was defined as 40 ng/mL with a minimum required dilution of 100.

Fig. 14A-14D summarize the effects of administration of aFXI-18623p IgG4 HC (S228P) (E1)/LC Kappa antibody on thrombus formation (FIGS. 14A, 14B), APTT (FIG. 14C), and PT (FIG. 14D). Table 8 summarizes the effect of aFXI-18623p IgG4 HC (S228P) (E1)/LC Kappa antibody on clot weight in a cynomolgus monkey model of AV shunt. Table 9 summarizes the effects of aFXI-18623p IgG4 HC (S228P) (E1)/LC Kappa antibody on aPTT and RT in a cynomolgus monkey model of AV shunt.

Table 8. Effect of aFXI-18623p IgG4 HC (S228P) (E1)/LC kappa antibody on clot weight __________in the cynomolgus monkey model of AV shunt__________

Antibody dose (mg/kg) Shunt #1 (Carrier) Shunt#2 (Antibody) % inhibition clot mass Antibody concentration (µg/ml) 1 772.0 10 100% 29.13 0, 1 957.0 10 100% 2.42 0.01 974.0 1007.0 -3% 0.17 0.03 927.0 935.0 -1% 0.54 0.04 909.0 887.0 2% 0.79 0.05 607.0 472.0 22% 0.91 0.05 710, 0 147.0 79% 1.03 0.05 688 66 90% 0.83

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Table 9. Effect of aFXI-18623p IgG4 HC (S228P) (E1)/LC kappa antibody on APTT and RT in the cynomolgus monkey _____________AV shunt model_____________

Antibody dose (mg/kg) % changes APTT % changes RT Antibody concentration (µg/ml) 1 143% 1% 29.13 0, 1 93% 1% 2.42 0.01 4% 3% 0.17 0.03 10% 1% 0.54 0.04 5% -2% 0.79 0.05 17% 2% 0.91 0.05 21% 0% 1.03 0.05 42% 3% 0.83

As shown in FIG. 14A, 14B and in table. 8, aFXI-18623p IgG4 HC (S228P) (E1)/LC Kappa antibody showed a dose- and plasma concentration-dependent reduction in clot weight with full efficacy (90-100% clot reduction) observed at plasma concentrations of [antibody] greater than 1 µg/ml (approximately 10 nM). As shown in FIG. 14C and in table. 9, the antibody showed a dose- and plasma concentration-dependent increase in aPTT. With a plasma concentration of 2.4 μg/ml (~17 nM), aFXI-18623p IgG4 HC (S228P)(E1)/LC Kappa antibody showed a 93% increase in aPTT, while 29 μg/ml (~200 nM) antibody aFXI-18623p IgG4 NS (S228P)(E1)/LC Kappa (at the highest dose tested) resulted in a 143% increase in aPTT. Unlike APTT, as shown in FIG. 14D and in table. 9, the PT changed by less than 10% among the antibody concentrations assessed, consistent with the selective effect of FXI inhibition on the intrinsic coagulation pathway.

Example 9 Patterned bleeding time model in the cynomolgus monkey

The bleeding propensity of the anti-FXI mAb aFXI-18623p IgG4 HC (S228P) (E1)/LC Kappa was characterized in vivo using the cynomolgus monkey model of patterned bleeding time, developed at the research laboratories of Merck, Sharp & Dohme Corp., Kenilworh, NJ USA and Palo Alto, CA USA. This model has previously been used to demonstrate significant increases in patterned bleeding time at multiple anatomical sites with triple antiplatelet therapy (Cai et al., Eur. J. Pharmacol. 758:107-114 (2015)).

To use this model, patterned bleeding times were determined using spring lancets on the buccal mucosa (inner lip), finger pad, and tail tip at various time points to induce bleeding.

Bleeding time study:

Bleeding time studies were performed in anesthetized cynomolgus monkeys as follows.

Each test site (buccal mucosa, finger pad, or tail tip) was examined to identify suitable incision sites to induce bleeding.

To induce bleeding, a spring lancet was placed tightly against the selected area of interest and activated to produce a uniform linear incision. The lancet specifications determined the incision dimensions.

Blood from the incision site was allowed to flow freely and observed for 30 continuous seconds after bleeding had stopped. This determines the bleeding time (BT). VT was recorded for each site. During the determination of VT, the incision site at the end of the tail was irrigated with warm, sterile lactated Ringer's solution, and the toe pad site was immersed in warm, sterile lactated Ringer's solution. Using lactated Ringer's solution improves the ability to see blood flow from these areas.

Study design:

Each study included three 30-minute pattern bleeding time (BT) tests in three study areas (see FIG. 15, study design). The first VT determined the initial bleeding. The second VT occurred 70 min after a 3-min IV infusion (4.17 ml/kg) of compound-free vehicle (20 mM sodium acetate, 9% sucrose, pH 5.5) (Treatment #1). The third VT VT occurred 70 min after a 3-min IV infusion (4.17 ml/kg) of compound-free vehicle or aFXI-18623p IgG4 HC (S228P)(E1)/LC Kappa (10 mg/kg) (Treatment #2). Bleeding was observed and bleeding time was recorded as described above. The time at which bleeding stopped was noted for each site. Blood samples were collected periodically to determine circulating plasma levels of aFXI-18623p IgG4 HC antibody (S228P) (E1)/LC Kappa, aPTT, and RT.

Each study animal had two study sessions. In Study Session #1, vehicle followed by vehicle constituted Treatment #1 and Treatment #2, respectively. In Study Session #2, vehicle followed by 10 mg/kg IV aFXI-18623p IgG4 HC (S228P) (E1)/LC Kappa constituted Treatment #1 and Treatment #2, respectively.

The time period of 70 minutes between the end of the test drug infusion and the start of the bleeding time assessment reflects the time in the AV shunt model for determining thrombus mass (shunt placement 30 minutes after treatment + 40 minutes of blood flow through the shunt). The study dose of 10 mg/kg IV aFXI18623p IgG4 HC (S228P)(E1)/LC Kappa was determined to achieve 10x the predicted human _Cmax for aFXI-18623p IgG4 HC (S228P)(E1)/LC Kappa based on PK modeling studies/ FD in primates described previously.

Coagulation biomarkers, activated partial thromboplastin time (aPTT) and prothrombin time (PT), as well as circulating levels of plasma aFXI-18623p IgG4 HC (S228P) (E1)/LC Kappa antibody were measured in blood samples collected throughout the experiment, as shown in Fig. 15 APTT and PT were measured from thawed (previously frozen to -80°C) citrated plasma collected from animals using a Sta Compact Max coagulation analyzer (Stago Diagnostic, Inc). The Stago analyzer measures clot formation time using an electromagnetic mechanical clot detection system. For the aPTT assay, 50 μl of plasma was mixed with 50 μl of ellagic acid mixture (aPTT-XL, Pacific Hemostasis; Fisher Diagnostics catalog no. 10-0402) in a cuvette, which was then incubated at 37°C for 3 min. 50 μl of 0.025 M calcium chloride (Sta-CaCl ₂ 0.025 M, Stago Diagnostic, Inc., cat. no. 00367) was added to the mixture to initiate clotting, and the clot formation time was measured. For RT analysis, 50 μl of plasma in a cuvette was incubated at 37°C for 4 min; the onset of clotting was initiated by adding 100 μl of thromboplastin reagent (Triniclot PT Excel, TCoag, Inc., cat. no. T1106).

A standard electrochemiluminescence-based hIgG4 immunoassay was used to quantify aFXI-18623p IgG4 HC (S228P) (E1)/LC kappa in rhesus monkey plasma. The assay was performed with biotinylated goat anti-huIgG(H+L) from Bethyl (cat. no. A80-319B) as the capture reagent, and sulfoTAG-tagged mouse anti-human IgG (Fc-specific) from Southern Biotech (cat. no. 9190 -01) as a detection reagent. This assay was qualitative and the lower limit of quantitation was determined to be 41 ng/mL with a minimum required dilution of 100.

Fig. 16A-16F summarize the effects of vehicle and 10 mg/kg IV aFXI-18623p IgG4 HC (S228P)(E1)/LC Kappa in six cynomolgus monkeys on patterned bleeding times at the buccal mucosa (FIGS. 16A, 16D), fingertip (Fig. 16B, 16E) and the end of the tail (Fig. 16C, 16F). Effects on bleeding time were assessed by comparing absolute bleeding time (left panels) and percent change in bleeding time (right panels), with vehicle-vehicle Treatments #1 and 2 in study session #1, and vehicle-αFXI-18623p IgG4 HC (S228P) (E1)/LC Kappa as Treatments #1 and 2 in Study Session #2. Comparisons of absolute bleeding time for vehicle versus aFXI-18623p IgG4 HC (S228P) (E1)/LC Kappa, as well as percent change in bleeding time for vehicle versus vehicle-αFXI-18623p IgG4 HC (S228P) (E1 )/LC Kappa showed no statistically significant changes in bleeding time at any of the sites tested with aFXI-18623p IgG4 HC (S228P) (E1)/LC Kappa at this test dose.

The plasma concentration for aFXI-18623p IgG4 HC (S228P) (E1)/LC Kappa achieved at the 10 mg/kg IV test dose in the cynomolgus monkey bleeding time study was 290.7 ± 17.2 (mean ± SEM ) µg/ml (~1938.2 nM). Plasma APTT values were 31.0±0.5 s at baseline compared to 71.3±1.6 s after IV 10 mg/kg aFXI-18623p IgG4 HC (S228P) (E1)/LC Kappa (2 ,3x magnification). Plasma RT values were 12.7 ± 0.1 s at baseline compared to 12.6 ± 0.1 s after IV 10 mg/kg aFXI-18623p IgG4 HC (S228P) (E1)/LC Kappa (no noticeable increase).

Example 10. Evaluation of pharmacokinetics (PK) and pharmacodynamics (PD) of aFXI-18623p IgG4 HC (S228P)(E1)/LC kappa after multiple intravenous administrations in rhesus monkeys

The PK-PD properties of aFXI-18623p IgG4 HC (S228P) (E1)/LC kappa were characterized in vivo in rhesus monkey. The objective was to evaluate PK properties and establish PK/PD after two weekly doses.

Study design. Rhesus macaques (four animals per dose group) were administered (i.v.) vehicle without compound (10 mM sodium acetate, pH 5.5, 7% Sucrose, 0.02% PS-80) or aFXI-18623p IgG4 NS ( S228P) (E1)/LC kappa with five dose levels of 0.1, 0.3, 1, 3 and 6 mg/kg. The duration of the study was 22 days, and 1.5 ml of blood was collected to determine drug levels and activated partial thromboplastin time (aPTT).

Coagulation biomarker (APTT) and circulating plasma levels of aFXI-18623p IgG4 HC (S228P)(E1)/LC were measured in blood samples collected throughout the experiment, as shown in Table. 10.

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Table 10. Sample Collection Schedule

Collection type Time FC Day -3; Day 0: before dose (- 1 hour) and 30 minutes, 3 hours, 6 hours, 24 hours (Day 1), 48 hours (Day 2), 96 hours (Day 4) Day 7: before dose and 1 hour, 6 hours, 24 hours (Day 8), 48 hours (Day 9), 96 hours (Day 11), 168 hours (Day 14), 264 hours (Day 18) and 528 hours ( Day 22) after the second dose FD (estimate aPTT) Day -3; Day 0: before dose (- 1 hour) and 30 minutes, 3 hours, 6 hours, 24 hours (Day 1), 48 hours (Day 2), 96 hours (Day 4) Day 7: before dose and 1 hour, 6 hours, 24 hours (Day 8), 48 hours (Day 9), 96 hours (Day 11), 168 hours (Day 14), 264 hours (Day 18) and 528 hours ( Day 22) after the second dose

APTT was measured from thawed (previously frozen to -80°C) citrated plasma collected from animals using a Sta-R Evolution coagulation analyzer (Stago Diagnostic, Inc).

The clotting analyzer measures clot formation time using an electromagnetic mechanical clot detection system. For the APTT assay, the analyzer mixes 50 μL of plasma with 50 μL of ellagic acid (APTT-XL, Pacific Hemostasis; Fisher Diagnostics catalog no. 10-0402) in a cuvette, which is then incubated at 37°C for 3 min. Then, 50 μl of 0.025 M calcium chloride (Sta-CaCl ₂ 0.025 M, Stago Diagnostic, Inc., cat. no. 00367) was added to the mixture to initiate clotting, and the clot formation time was measured.

A standard electrochemiluminescence-based hIgG4 immunoassay was used to quantify aFXI-Ι 8623p IgG4 HC (S228P)(E1)/LC kappa in rhesus monkey plasma. The assay was performed with biotinylated goat anti-huIgG(H+L) from Bethyl (cat. no. A80-319B) as the capture reagent, and sulfoTAG-tagged mouse anti-huIgG (Fc-specific) from Southern Biotech (cat. no. 9190- 01) as a detection reagent. This assay was qualitative and the lower limit of quantitation was determined to be 41 ng/mL with a minimum required dilution of 100.

Plasma concentration-time data for individual animals for aFXI-Ι 8623p IgG4 HC (S228P) (E1)/LC kappa were analyzed using non-compartmental (NCA) methods (Gabrielsson and Weiner, 2000). All PK parameters were determined or calculated using Phoenix 32 WinNonlin 6.3 (version 6.3.0.395, Certara L.P. St. Louis, MO, 2012). Non-compartmental analysis used Model 201 (IV). All concentration data and PK parameters were rounded to three significant figures. Samples with concentration values below the lower limit of detection (< LLOQ) were excluded from PK analysis and calculation of mean data. For plotting, values < LLOQ were set to 1/2 the minimum recorded concentration for concentration-time plots for an individual animal.

The _Emax sigmoidal response (PK/PD) model was used to characterize the relationship between exposure and aPTT using GraphPad Prism version 7.00 (GraphPad Software Inc). In the model, the E _max value corresponds to the maximum increase in aPTT achieved from the initial level, and the EC ₅₀ value corresponds to the half-maximal effective concentration. Variability is described as the 95% confidence interval (CI) for the _EC50 value provided by the software.

results

Individual concentration-time profiles for aFXI-Ι8623p IgG4 HC (S228P) (E1)/LC kappa are shown in FIG. 17A. Nonlinearity was observed for all PK parameters. Mean clearance values decreased from approximately 8 mL/kg-day for the lowest dose tested (0.1 mg/kg) to approximately 4 mL/kg-day for the highest dose tested (6 mg/kg). APTT concentration-time profiles are shown in Fig. 17V. A dose-dependent increase in aPTT was observed. The relationship between plasma concentrations of aFXI-18623p IgG4 HC (S228P) (E1)/LC kappa and aPTT was best described by the sigmoid E _max model, which adequately described this relationship. The calculated EC ₅₀ value for aFXI18623p IgG4 HC (S228P) (E1)/LC kappa was approximately 3.6 μg/ml.

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Sequence table

SEQ ID NO: Description Subsequence 1 CDR1 severe aFXI-chains 18611r and aFXI18611 YSISSGYFWG 2 CDR2 severe aFXI-chains 18611r and aFXI18611 SILHSGVTYYNPSLKS 3 CDR3 severe aFXI-chains 18611r ARDRTTVSMIEYFQH 4 CDR3 severe chains aFXI-18611 ARDRTTVSLIEYFQH 5 CDR1 light aFXI-chains 18611r and αΕΧΙ18611 QASQDISNYLN 6 CDR2 easy aFXI-chains 18611r and αΕΧΙ18611 DASNLET 7 CDR3 easy aFXI-chains 18611r and αΕΧΙ18611 QQFHLLPIT 8 CDR1 severe aFXI-chains 18623rub GSIYSGAYYWS 9 CDR2 severe chains αΕΧΙ- SIHYSGLTYYNPSLKS

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18623p 10 CDR3 severe aFXI-chains 18623rub ARDDVDDSSGDEHYGMDV eleven CDR1 light aFXI-chains 18623rub RASQGIDSWLA 12 CDR2 easy aFXI-chains 18623rub AASSLQS 13 CDR3 easy aFXI-chains 18623rub QQYHIVPIT 14 Light chain leader sequence A MSVPTQVLGLLLLWLTDARC 15 Leader sequence in the heavy chain MEWSWVFLFFLSVTTGVHS 16 Constant domain heavy IgG4 chains person: (S228P) S in position 108 replaced by P ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPP CPAPEFLGGPSVFLFPPKPKDTLMLSRTPEVTCVVV DVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTY RVVSVL TVLHQDWLNGKEYKCKVSNKGLPSSLEKTL SKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLS LGK 17 Constant domain heavy IgG4 chains ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPP

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human : (S228P)S in position 108 substituted by P; C-terminal K absent CPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSLEKTL SKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLS LG 18 Constant domain heavy IgGl chains person ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SSSLGTQTYLCNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMLSRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVV SVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDLAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK 19 Constant domain heavy IgGl chains human C-terminal K absent ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SSSLGTQTYLCNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMLSRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVV SVLTVLHQDWLNGKEYKCKVSNKALPAPLE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDLAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPG 20 Human kappa LC constant domain RTVAAPSVFLFPPSDEQLKSGTASVVCLLNNFYPRE AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 21 Variable region NS aFXI-18611p; (QI) (M105) OVOLQESGPGLVKPSETLSLTCAVSGYSISSGYFWG WIRQPPGKGLEWIGSILHSGVTYYNPSLKSRVTISV DT S KNQ ES LKL S SVTAADTAVYYCARDRTTVSMIEY FQHWGOGTLVTVSS

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22 Variable EVQLQESGPGLVKPSETLSLTCAVSGYSISSGYFWG WIRQP P GKGLEWIGSILHSGVTYYNPSLKSRVTISV DT S KNQ ES LKL S SVTAADTAVYYCARDRTTVSMIEY FQHWGOGTLVTVSS NS area aFXI-18611p; (El) (M105) 23 Variable region NS aFXI-18611; (QI) (L105) OVOLOESGPGLVKPSETLSLTCAVSGYSISSGYFWG WIRQP R GKGLEWIGSILHSGVTYYNPSLKSRVTISV DTSKNQFSLKLSSVTAADTAVYYCAR DRTTVSLIEYFQHWGOGTLVTVSS 24 Variable region NS aFXI-18611; (El) (L105) EVOLOESGPGLVKPSETLSLTCAVSGYSISSGYFWG WIRQP P GKGLEWIGSILHSGVTYYNPSLKSRVTISV DTSKNQFSLKLSSVTAADTAVYYCAR DRTTVSLIEYFQHWGOGTLVTVSS 25 Variable region LC aFXI-18611p and aFXI-18611 DIQMTQSPSSL SASVGDRVTIT CQASQDISNYLNWY QQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFT FTISSLOPEDIATYYCQQFHLLPITFGGGTKVEIK 26 LC mouthguard aFXI- 18611r and aFXI18611 DIQMTQSPSSL SASVGDRVTIT CQASQDISNYLNWY QQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFT FTISSLQPEDIATYYCQQFHLLPITFGGGTKVEIKR TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC 27 DNA encoding LC kappa aFXI18611р and aFXI 18611 GACAT C CAGAT GAC C CAGAG C C C TAG CAG CCT GAG C GCCAGCGTGGGCGACAGAGTGACCATCACCTGTCAA GCCTCCCAGGACATCTCCAACTACCTGAACTGGTAC CAGCAAAGCCCGGCAAGGCTCCCAAGCTGCTGATC TACGACGCCTCCAACCTGGAGACCGGCGTGCCTAGC AGATTTAGCGGCAGCGGCTCCGGCACAGACTTCACC TTCACCATCA GCTCCCTGCAGCCCGAGGACATTGCC ACCTACTACTGCCAGCAGTTTCACCTGCTGCCTATC ACCTTCGGCGGCGGCACCAAGGTGGAGATCAAAAGG ACCGTCGCCGCCCCTAGCGTGTTCATCTTCCCCCCT AGCGACGAGCAGCTCAAGTCCGGCACCGCCAGCGTG GTGTGTCTGCTCAACAACTTCTACCCCAGGGAGGCC

- 45 043303

AAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGC GGCAACAGCCAGGAGAGCGTGACAGAACAGGACAGC AAG GAT T S CASATACAG S S T GAG STS SAS S T GAC S CTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTAC GCCTGTGAGGTGACACACCAGGGCCTCAGCTCCCCC GT GACCAAGAGC T T CAACAGAGGCGAAT GC T GA 28 Variable region NS αΕΧΙ-18623ρ; (Q1) QVQLQESGВGLVKВSQТLSЛТСTVSGGSIYSGAYYW S WIRQHBGKGLEWIGSIHYSGLTYYNBSLKSRVTIS VDTSKNOFSLKLSSVTAADTAVYYCARDVDDSSGDE HYGMDVWGOGTTVTVSS 29 Variable region NS aFXI-18623p; (El) EVOLOESGBGLVKBSOTLSLTCTVSGGSIYSGAYYW S WIRQHBGKGLEWIGSIHYSGLTYYNBSLKSRVTIS VDTSKNOFSLKLSSVTAADTAVYYCARDVDDSSGDE HYGMDVWGOGTTVTVSS thirty Variable region LC aFXI-18623p DIQMTQS BS SVSASVGDRVTITCRASQGIDSWLAWY OQKBGKABKLLIYAASSLQSGVBSRFSGSGSGTDET LTISSLOPEDFATYYCOQYHIVPITFGGGTKVEIK 31 LC mouthguard aFXI- 18623rub DIQMTQS BS SVSASVGDRVTITCRASQGIDSWLAWY OQKBGKABKLLIYAASSLQSGVBSRFSGSGSGTDET LTISSLOPEDFATYYCQQYHIVPITFGGGTKVEIKR TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC 32 DNA encoding LC kappa αΕΧΙ18623р GACAT C CAGAT GAC C CAGAG C C C TAG CAG C G T GAG C GCCAGCGTGGGCGATAGGGTGACCATCACCTGCAGA GCCTCCCAGGGCATCGACAGCTGGCTGGCCTGGTAC CAGCAAAGCCCGGCAAGGCCCCTAAGCTGCTGATC TACGCCGCTAGCAGCCTGCAGAGCGGCGTGCCTAGC AGGTTCAGCGGAAGCGGCAGCGGCACCGACTTCACA CTGAC CATCAGCAGCCTGCAACCTGAGGACTTCGCC AC WITH TAC TAC T G WITH CAG CAG TAT CACAT CGTGCCCATC ACCTTCGGCGGCGGAACCAAGGTGGAGATTAAGAGG ACCGTGGCCGCCCCCAGCGTGTTTATCTTTCCCCCCC AGCGATGAGCAGCTGAAGAGCGGAACCGCCAGCGTG GTGTGCCTGCTGAACAACTTCTACCCCAGAGAGGCC

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33 34 HC IgG4 aFXI18611p (S228P) (Ql) (M105) DNA encoding HC IgG4 aFXI18611p (S228P)(Ql) (Ml 05) ; xxx= CAG or CAA (Q) AAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCC GGAAACAGCCAGGAGAGCGTGACCGAGCAGGATTCC AAGGATAGCACC TACAGCC T GAGCAGCACCC T GACA CTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTAC GCCTGTGAGGTGACCCATCAGGGCCTGAGCAGCCCT GTGACCAAGAGCTTCAACAGGGGCGAGTGCTGA OVOLOESG PGLVKPSETLSLTCAVSGYSISSGYFWG WIRQP P GKGLEWIGSILHSGVTYYNPSLKSRVTISV DT S KNQ FS LKL S SVTAADTAVYYCARDRTTVSMIEY FQHWGOGTLVTVSS ASTKGPSVFPLAPCSRSTSEST AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGT KTYTCNVDHKPSNTKV DKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHN AKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEM TKNQVSLTCLVKGF A TCGGTTCTATCCTGCACTCCGGCGTGACATACTAT AACCCTAGCCTGAAGAGCAGGGTGACCATCTCCGTG GATACCAGCAAGAAT CAGT T CAGCC T GAAGC T CAGC AGCGTGACCGCCGCCGATACCGCTGTGTACTACTGC G C CAGAGACAG GAC CAC CGTCTCCATGATC GAG TAC TTCCAGCACTGGGGCCAAGGCACCCTGGTCACCGTG TCCTCCGCCTCCACCAAGGGCCCTAGCGTGTTTCCT CTGGCCCCCTGCTCCAGATCCACAAGCGAGAGCACC GCTGCCCTGGGCTGTCTGGTCAAGGACTACTTCCCC GAGCCCGTGACAGTGTCCTGGAACAGCGGCGCCCTG ACAAGCGGCGTCCATA CATTCCCCGCCGTGCTGCAG TCCAGCGGACTGTATAGCCTGAGCTCCGTGGTGACC

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GTGCCTTCCAGCAGCCTGGGAACCAAGACATATACC TGCAACGTGGACCATAAGCCCAGCAACACAAAAGTC GACAAGAGGGTGGAGAGCAAGTACGGACCCCCTTGT CCCCCTTGTCCTGCTCCCGAGTTCCTCGGCGGACCT AGCGTGTTCCTGTTTCCTCCCAAGCCCAAGGATACC CTGATGATCAGCAGGACCCCTGAGGTCACCTGCGTG GTGGTCGACGTGTCCCAGGA GGACCCTGAGGTCCAG TTTAACTGGTACGTGGACGGAGTGGAGGTGCACAAC G C CAAGAC CAAG C C CAGAGAG GAG CAG T T CAAT T C C ACCTACAGGGTGGTGAGCGTCCTGACCGTGCTGCAC CAGGACTGGCTGAATGGAAAGGAGTACAAATGCAAG GTCTCCAACAAGGGCCTCCCTAGCAGCATCGAGAAG ACCATTCTCCAAGGCCAAGGGCCAGCCTAGGGAGC CC CAGGTGTACACCCTGCCTCCTAGCCAGGAGGAAATG ACCAAGAACCAGGTGTCCCTGACATGCCTGGTGAAG GGCTTCTATCCTAGCGACATCGCCGTGGAGTGGGAG AGCAATGGCCAGCCCGAGAATAACTACAAGACCCACC CCCCCTGTGCTCGATAGCGACGGCAGCTTCTTTCTG TACAGCAGGCTGACCGTGGACAAGAGCAGGTGGCAA GAGGGCAACGTGTTTA GCTGCTCCGTCATGCACGAG GCCCTGCATAACCACTACACCCAAAAATCCCTGTCC CTGTCCCTGGGCAAGTGA 35 NS IgG4 aFXI18611p (S228P) (El) (M105) EVQLQESGPGLVKPSETLSLTCAVSGYSISSGYFWG WIRQPPGKGLEWIGSILHSGVTYYNPSLKSRVTISV DT S KNQ FS LKL S SVTAADTAVYYCARDRTTVSMIEY FQHWGOGTLVTVSS ASTKGPSVFPLAPCSRSTSEST AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSV VTVPSSSLGTKTYTCNVDHKPSNTKV DKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHN AKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEM TKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLGK

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36 DNA encoding NS IgG4 αΚΧΙ18611р (S228P); (El) (M105) xxx=GAA or GAG (E) xxxGTCCAGCTGCAGGAGAGCGGCCCTGGCCTGGTG AAGCCTAGCGAGACACTGTCCCTGACCTGCGCCGTG AGCGGCTACAGCATCTCCAGCGGCTATTTCTGGGGA TGGATCAGACAGCCCCCTGGCAAGGGCCTGGAATGG ATCGGTTCTATCCTGCACTCCGGCGTGACATACTAT AACCCTAGCCTGAAGAGCAGGGTGACCATCTCCGTG GATACCAGCAAGAAT CAG T T CAGCC T GAAGC T CAGC AGCGTGACCGCCGCCGATACCGCTGTGTACTACTGC G C CAGAGACAG GAC CAC CGTCTCCATGATC GAG TAC TTCCAGCACTGGGGCCAAGGCACCCTGGTCACCGTG TCCTCCGCCTCCACCAAGGGCCCTAGCGTGTTTCCT CTGGCCCCCTGCTCCAGATCCACAAGCGAGAGCACC GCTGCCCTGGGCTGTCTGGTCAAGGACT ACTTCCCC GAGCCCGTGACAGTGTCCTGGAACAGCGGCGCCCTG ACAAGCGGCGTCCATACATTCCCCGCCGTGCTGCAG TCCAGCGGACTGTATAGCCTGAGCTCCGTGGTGACC GTGCCTTCCAGCAGCCTGGGAACCAAGACATATACC TGCAACGTGGACCATAAGCCCAGCAACACAAAAGTC GACAAGAGGGTGGAGAGCAAGTACGGACCCCCTTGT CCCCCTTGTCCTGC TCCCGAGTTCCTCGGCGGACCT AGCGTGTTCCTGTTTCCTCCCAAGCCCAAGGATACC CTGATGATCAGCAGGACCCCTGAGGTCACCTGCGTG GTGGTCGACGTGTCCCAGGAGGACCCTGAGGTCCAG TTTAACTGGTACGTGGACGGAGTGGAGGTGCACAAC G C CAAGAC CAAG C C CAGAGAG GAG CAG T T CAAT TCC ACCTACAGGGTGGTGAGCGTCCTGACCGTGCTGCAC CAGGACTGGCTGAATGGAAAGGAGTACAAATGCAAG GTCTCCAACAAGGGCCTCCCTAGCAGCATCGAGAAG ACCATCTCAAGGCCAAGGGCCAGCCTAGGGAGCCC CAGGTGTACACCCTGCCTCCTAGCCAGGAGGAAATG ACCAAGAACCAGGTGTCCCTGACATGCCTGGTGAAG GGCTTCTATCCTAGCGACATCGCCGTG GAGTGGGAG AGCAATGGCCAGCCCGAGAATAACTACAAGACCACC CCCCCTGTGCTCGATAGCGACGGCAGCTTCTTTCTG TACAGCAGGCTGACCGTGGACAAGAGCAGGTGGCAA

- 49 043303

GAGGGCAACGTGTTTAGCTGCTCCGTCATGCACGAG GCCCTGCATAACCACTACACCCAAAAATCCCTGTCC CTGTCCCTGGGCAAGTGA 37 NS IgG4 aFXI- 18611 (S228P) (QD (L105) OVOLOESGPGLVKPSETLSLTCAVSGYSISSGYFWG WIRQPPGKGLEWIGSILHSGVTYYNPSLKSRVTISV DT S KNQ FS LKL S SVTAADTAVYYCARDRTTVSLIEY FQHWGOGTLVTVSS ASTKGPSVFPLAPCSRSTSEST AALGCLVKDYFPEPVTVSWESGALTSGVHTFPAVLQ SSGLYSLSSVVT VPSSSLGTKTYTCNVDHKPSNTKV DKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHN AKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEM TKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLGK 38 DNA encoding NS IgG4 aFXI18611 (S228P); (QD (L105) xxx=CAG or CAA(Q) xxxGTCCAGCTGCAGGAGAGCGGCCCTGGACTCGTG AAGCCCTCCGAAACCCTGAGCCTCACATGCGCCGTC TCCGGATACAGCATCAGCAGCGGATACTTCTGGGGC TGGATCAGACAGCCCCCCGGCAAAGGCCTGGAGTGG ATCGGTTCTATTCTC CACAG C G G C G T GACATAC TAG AACCCCTCCCTGAAGAGCAGGGTGACCATCAGCGTG GACAC C T C CAAGAAC CAG TTTTCCCT CAAG C T GAG C AGCGTGACCGCCGCTGACACAGCCGTGTATTACTGC G C CAG G GAC AG GAC C AC CGTGTCCCTGATT GAG TAG TTCCAGCATTGGGGCCAGGGCACACTGGTGACCGTC AGCAGCGCCAGCACCAAGGGCCCTTCCGTCTTCCCT CTGGCCCCTTGCAGCAGAAGCACCTCCGAGTCCACA GCCGCCCTG GGATGCCTCGTGAAGGATTACTTCCCC GAGCCCGTCACAGTCTCCTGGAACTCCGGCGCTCTG ACCAGCGGAGTGCACACCTTCCCCGCCGTGCTGCAA AGCAGCGGCCTGTACAGCCTGTCCAGCGTGGTCACC GTGCCTTCCTCCAGCCTGGGCACCAAGACCTACACA T G CAAC G T G GAC CACAAG C C T T WITH CAACAC CAAG G T G GACAAGAGAGTGGAAAGCAAGTACGGCCCCCCCTGC

- 50 043303

CCCCCTTGTCCTGCCCCCGAGTTTCTGGGAGGACCC TCCGTGTTCCTCTTTCCTCCCAAGCCTAAGGACACC CTGATGATCTCCAGGACCCCCGAAGTGACCTGCGTG GTCGTGGACGTGTCCCAGGAGGACCCTGAGGTGCAG TTTAACTGGTACGTGGACGGCGTGGAGGTGCACAAC GCCAAGACCAAGCCCAGGGAGGAGCAGTTCAATAGC ACCTACAGGTGGTGTC CGTGCTGACCGTGCTGCAC CAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAA GTCAGCAACAAGGGCCTGCCCTCCTCCATCGAGAAG ACCATTAGCAAGGCCAAGGGCCAGCCTAGGGAGCCT CAGGTGTACACCCTGCCCCCCAGCCAGGAGGAGATG ACCAAGAACCAGGTGTCCCTGACCTGCCTGGTCAAG GGATTTTACCCCAGCGACATCGCTGTGGAATGGGAG AG CA AT GGC CAG C C GAGAACAAC TACAAGAC CAC C CCTCCCGTGCTCGATTCCGACGGCAGCTTTTTCCTG TACAGCAGGCTGACCGTGGATAAGAGCAGGTGGCAG GAAGGCAACGTGTTCTCCTGTTCCGTGATGCATGAG G C C C T G CACAAC CAC TACACACAGAAGAG C C T G T C C CTGTCCCTGGGCAAGTGA 39 NS IgG4 aFXI- 18611 (S228P) (El) (L105) EVOLQESGPGLVKPSETLSLTCAVSGYSISSGYFWG WIRQPPGKGLEWIGSILHSGVTYYNPSLKSRVTISV DT S KNQ FS LKL S SVTAADTAVYYCARDRTTVSLIEY FQHWGOGTLVTVSS ASTKGPSVFPLAPCSRSTSEST AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVT VPSSSLGTKTYTCNVDHKPSNTKV DKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHN AKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEM TKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLGK 40 DNA encoding NS IgG4 aFXI18611 (S228P) xxxGTCCAGCTGCAGGAGAGCGGCCCTGGACTCGTG AAGCCCTCCGAAACCCTGAGCCTCACATGCGCCGTC TCCGGATACAGCATCAGCAGCGGATACTTCTGGGGC

- 51 043303

(Ql) (L105) xxx=GAA or GAG (E) TGGATCAGACAGCCCCCCGGCAAAGGCCTGGAGTGG ATCGGTTCTATTCTC GAGAG C G G C G T GACATAC TAC AACCCCTCCCTGAAGAGCAGGGTGACCATCAGCGTG GACAC STS CAAGAAC CAG TTTTCCCT CAAG C T GAG C AGCGTGACCGCCGCTGACACAGCCGTGTATTACTGC G C CAG G GACAG GAC GAG CGTGTCCCTGA TT GAG TAC TTCCAGCATTGGGGCCAGGGCACACTGGTGACCGTC AGCAGCGCCAGCACCAAGGGCCCTTCCGTCTTCCCT CTGGCCCCTTGCAGCAGAAGCACCTCCGAGTCCACA GCCGCCCTGGGATGCCTCGTGAAGGATTACTTCCCC GAGCCCGTCACAGTCTCCTGGAACTCCGGCGCTCTG ACCAGCGGAGTGCACACCTTCCCCGCCGTGCTGCAA AGCAGCGGCC TGTACAGCCTGTCCAGCGTGGTCACC GTGCCTTCCTCCAGCCTGGGCACCAAGACCTACACA T G CAAC GTG GAC CACAAGС C T T C CAACACCAAGGTG GACAAGAGAGTGGAAAGCAAGTACGGCCCCCCCTGC CCCCCTTGTCCTGCCCCCGAGTTTCTGGGAGGACCC TCCGTGTTCCTCTTTCCTCCCAAGCCTAAGGACACC CTGATGATCTCCAGGAC CCCCGAAGTGACCTGCGTG GTCGTGGACGTGTCCCAGGAGGACCCTGAGGTGCAG TTTAACTGGTACGTGGACGGCGTGGAGGTGCACAAC GCCAAGACCAAGCCCAGGGAGGAGCAGTTCAATAGC ACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCAC CAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAA GTCAGCAACAAGGGCCTGCCCTCCTCCATCGAGAAG ACCATTAGCAAGGCCAAGGGCCAGCCTAGGGAGCCT CAGGTGTACACCCTGCCCCCCAGCCAGGAGGAGATG ACCAAGAACCAGGTGTCCCTGACCTGC CTGGTCAAG GGATTTTACCCCAGCGACATCGCTGTGGAATGGGAG AG СAAT GGC CAG CCC GAGAACAAC TACAAGACСACC CCTCCCGTGCTCGATTCCGACGGCAGCTTTTTCCTG TACAGCAGGCTGACCGTGGATAAGAGCAGGTGGCAG GAAGGCAACGTGTTCTCCTGTTCCGTGATGCATGAG G C C C T G CACAACСAC TACACACAGA AGAGCC T GT CC CTGTCCCTGGGCAAGTGA

- 52 043303

41 HC-IgG4 αΕΧΙ- 18623p (S228P ( (QI) QVQLQESGPGLVKPSQTLSLTCTVSGGSIYSGAYYW SWIRQHPGKGLEWIGSIHYSGLTYYNPSLKSRVTIS VDTSKNQFSLKLSSVTAADTAVYYCARDVDDSSGDE HYGMDVWGQGT TVTVS S ASTKGPSVFPLAPCSRSTS ESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSG LYSLSSVVTVPSSSLGTKTYTCNVDHKPSN TKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQ EEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV MHEALHNHYTQKSLSLSLGK 42 DNA encoding pHC-IgG4 αΕΧΙ- 18623 (S228P((QI) xxx=CAG or CAA(Q) xxxGTCCAGCTGCAGGAATCCGGACCCGGCCTGGTG AAGCCTAGCCAGACCCTGAGCCTGACCTGTACCGTG TCCGGCGGAAGCATCTATTCCGGCGCCTACTACTGG TCCTGGATTAGGCAGCACCCCGGCAAGGGCCTGGAA TGGATCGGCTCCATCCACTACAGCGGCCTGACCTAT TACAACCCCTCCCTGAAGTCCAGGGTGACCATCAGC GTC GACACAAG CAAGAAC CAG TTCTCCCT CAAG C T G AGCAGCGTGACCGCCGCCGACACCGCCGTGTATTAT T G C G C CAGAGAC GTG GAC GAC T C C T C C G GAGAC GAG CACTACGGCATGGACGTCTGGGGCCAGGGCACAACA GTGACAGTGAGCAGCGCCAGCACCAAAGGACCCTCC GTCTTCCCTCTGGCCCCTTGCTCCAGGAGCACAAGC GAAAGCACAGCCGCCCTGGG CTGCCTGGTGAAGGAC TACTTTCCCGAGCCCGTGACCGTGAGCTGGAATAGC GGAGCCCTCACCTCCGGAGTCCACACATTTCCCGCC GTCCTGCAGAGCAGCGGCCTGTACTCCCTGAGCTCC GTGGTGACCGTGCCTTCCTCCAGCCTGGGCACCAAG AC WITH TACAC WITH T G CAAC GTG GAC CACAAG WITH WITH TAG CAAT ACCAAGGTGGACAAGAGGGTGGAATCCAAGTACGGC CCCCCTTGCCCTCCTTGTCCTGCCCCCGAATTTCTG GGCGGCCCTTCCGTGTTCCTGTTCCCTCCCAAGCCC AAG GATAC C C T GAT GAT CAG CAG GAC C C C T GAG GTG

- 53 043303

ACCTGTGTGGTGGTGGACGTGAGCCAGGAGGACCCC GAGGTGCAGTTCAACTGGTACGTGGATGGCGTGGAA GTGCACAATGCCAAGACAAAGCCCAGGGAGGAGCAG TTCAATAGCACCTACAGGGTGGTCAGCGTGCTCACA GTGCTGCACCAGGACTGGCTGAACGGAAAGGAGTAC AAGTGCAAAGTGTCCAACAAGGGCCTGCCCTCCTCC ATCGAAAAGACCATCTCCAA GGCCAAAGGCCAGCCC AGGGAGCCCCAAGTGTATACCCTCCCCCCTAGCCAG GAGGAAATGACСAAAAAACCAGGTCTCCCTGACCTGT CTGGTGAAGGGCTTCTATCCCAGCGACATCGCTGTG GAGTGGGAGAGCAACGGCCAACCCGAGAACAACTAT AAGACCACACCCCCCGTCCTGGACTCCGATGGCTCC TTCTTCCTGTACAGCAGGCTGACCGTCGACAAGTC C AGGTGGCAGGAAGGAAACGTGTTCTCCTGTAGCGTC ATG CAC GAG G C C C T G CACAACСAC TATACCCAGAAG TCCCTGTCCCTGAGCCTGGGCAAGTGA 43 HC-IgG4 aFXI18623p (S228P( (El) EVQLQESGPGLVKPSQTLSLTCTVSGGSIYSGAYYW SWIRQHPGKGLEWIGSIHYSGLTYYNPSLKSRVTIS VDTSKNQFSLKLSSVTAADTAVYYCARDVDDSSGDE HYGMDVWGQGT TVTVS S ASTKGPSVFPLAPCSRSTS ESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLY SLSSVVTVPSSSLGTKTYTCNVDHKPSN TKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQ EEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV MHEALHNHYTQKSLSLSLGK 44 DNA encoding HC IgG4 aFXI18623p (S228P((El) xxx=GAA or GAG (E) xxxGTCCAGCTGCAGGAATCCGGACCCGGCCTGGTG AAGCCTAGCCAGACCCTGAGCCTGACCTGTACCGTG TCCGGCGGAAGCATCTATTCCGGCGCCTACTACTGG TCCTGGATTAGGCAGCACCCCGGCAAGGGCCTGGAA TGGATCGGCTCCATCCACTACAGCGGCCTGACCTAT TACAACCCCTCCCTGAAGTCCAGGGTGACCATCAGC

- 54 043303

GTC GACACAAGCAAGAAC CAG TTCTCCCT CAAGС T G AGCAGCGTGACCGCCGCCGACACCGCCGTGTATTAT T G C G C C AGAGAC GTG GAC GAC T C C T C G GAGAC GAG CACTACGGCATGGACGTCTGGGGCCAGGGCACAACA GTGACAGTGAGCAGCGCCAGCACCAAAGGACCCTCC GTCTTCCCTCTGGCCCCTTGCTCCAGGAGCAC AAGC GAAAGCACAGCCGCCCTGGGCTGCCTGGTGAAGGAC TACTTTCCCGAGCCCGTGACCGTGAGCTGGAATAGC GGAGCCCTCACCTCCGGAGTCCACACATTTCCCGCC GTCCTGCAGAGCAGCGGCCTGTACTCCCTGAGCTCC GTGGTGACCGTGCCTTCCTCCAGCCTGGGCACCAAG AC WITH TACAC WITH T G CAAC GTG GAC CACAAGС WITH TAGCAAT ACCAAGGTGGACAAG AGGGTGGAATCCAAGTACGGC CCCCCTTGCCCTCCTTGTCCTGCCCCCGAATTTCTG GGCGGCCCTTCCGTGTTCCTGTTCCCTCCCAAGCCC AAG GATAC C C T GAT GAT CAG CAG GAC C C C T GAG GTG ACCTGTGTGGTGGTGGACGTGAGCCAGGAGGACCCC GAGGTGCAGTTCAACTGGTACGTGGATGGCGTGGAA GTGCACAATGCCAAGACAAAGCCCAGGGAGGAGCAG TTCAATAGCACCTACAGGGTGGTCAGCGTGCTCACA GTGCTGCACCAGGACTGGCTGAACGGAAAGGAGTAC AAGTGCAAAGTGTCCAACAAGG GCCTGCCCTCCTCC ATCGAAAAGACCATCTCCAAGGCCAAAGGCCAGCCC AGGGAGCCCCAAGTGTATACCCTCCCCCTAGCCAG GAG GAAAT GAC CAAAAAC CAG G T C T C C C T GAC C T G T CTGGTGAAGGGCTTCTATCCCAGCGACATCGCTGTG GAGTGGGAGAGCAACGGCCAACCCGAGAACAACTAT AAGACCACACCCCCCGTCCTGGACTCCGATGG CTCC TTCTTCCTGTACAGCAGGCTGACCGTCGACAAGTCC AGGTGGCAGGAAGGAAACGTGTTCTCCTGTAGCGTC ATG CAC GAG G C C C T G CACAACСAC TATACCCAGAAG TCCCTGTCCCTGAGCCTGGGCAAGTGA 45 NS IgGl aFXI- 18611p(QI) (M105) QVQLQESGPGLVKPSETLSLTCAVSGYSISSGYFWG WIRQPPGKGLEWIGSILHSGVTYYNPSLKSRVTISV DT S KNQ FS LKL S SVTAADTAVYYCARDRTTVSMIEY

- 55 043303

FQWGOGTLMTMSSASTKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTEMPERTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK 46 DNA encoding NS IgGl aFXI18611p (QI) (Ml 05) xxx= CAG or CAA (Q) xxxGTCCAGCTGCAGGAGAGCGGCCCTGGCCTGGTG AAGCCTAGCGAGACACTGTCCCTGACCTGCGCCGTG AGCGGCTACAGCATCTCCAGCGGCTATTTCTGGGGA TGGATCAGACAGCCCCCTGGCAAGGGCCTGGAATGG ATCGGTTCTATCCTGCACTCCGGCGTGACATACTAT AACCCTAGCCTGAAGAGCAGGGTGACCATCTCCGTG GATACCAGCAAGAAT CAG T T CAGCC T GAAGC T CAGC AGCGTGACCGCCGCCGATACCGCTGTGTACTACTGC G C CAGAGACAG GAC CAC CGTCTCCATGATC GAG TAC TTCCAGCACTGGGGCCAAGGCACCCTGGTCACCGTG TCCTCCGSTAGCACAAAAGGACCAAGCGTGTTTCCA CTGGCACCTAGCAGCAAATCCACCAGCGGCGGAACA GCAGCCCTCGGGTGCCTGGTGAAG GATTACTTCCCT GAGCCAGTCACAGTGTCCTGGAACTCCGGAGCCCTG ACATCCGGCGTGCACACCTTCCCCGCTGTGCTGCAA TCCAGCGGACTGTATAGCCTCAGCTCCGTCGTGACA GTCCCTTCCAGCAGCCTGGGCACACAGACTTACATT T G CAAC G T GAAC CACAAAC C T T C CAACAC TAAG GTG GACAAAAAG GTG GAAC C CΑΑ ΆΤ C C T G T GATAAGAC C CATACATGCCCACCTTGTCCCGCTCCTGAGCTGCTG GGGGGACCTTCCGTCTTTCTGTTTCCTCCAAAACCA AAAGACACAC T CAT GAT CAG C C G GAC C C C GAAG T C ACCTGTGTGGTGGTGGACGTCAGCCACGAAGATCCA GAGGTCAAGTTCAATTGGTACGTGGATGGAGTGGAA GTC CACAAC G CAAAAAC CAAAC C TAGAGAAGAACAG

- 56 043303

TACAATAG CACATACAG GGTGGTGTCCGTCCT GACA GTGCTCCACCAGGACTGGCTCAATGGCAAAGAGTAT AAGTGCAAGGTGAGCAACAAGGCCCTGCCTGCACCA AT T GAGAAAACAAT TAGCAAGGCAAAGGGGCAGCCA CGGGAACCCCAGGTGTATACCCTGCCCCCAAGCCGG GAT GAAC T GAC C AAAAAC CAG G T CAG CCT GAC AT G C CTGGTGAAAGGGTTTTACCAAGCGATATTGCCGTC GAGTGGGAGAGCAACGGACAGCCAGAAAACAATTAC AAAACCACCCCACCTGTGCTGGACTCCGATGGGAGC TTTTTCCTG TACAG CAAG C T CACAG T G GACAAG TCC AGATGGCAACAGGGCAACGTGTTTTCCTGCTCCGTG ATG CAC GAG G C C T C CACAAC CAC TATACACAAAAG TCCC TCTCCCTCAGCCCAGGAAAGTGA 47 NS IgGl αΕΧΙ- 18611p (El) (M105) EVQLQESGPGLVKPSETLSLTCAVSGYSISSGYFWG WIRQPPGKGLEWIGSILHSGVTYYNPSLKSRVTISV DT S KNQ ES LKL S SVTAADTAVYYCARDRTTVSMIEY FQRWGOGTLVTVSSASTKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTEMPERTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPEEKTESKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK 48 DNA encoding NS IgGl αΕΧΙ18611p (QI) (Ml 05) xxx=GAA or GAG(E) xxxGTCCAGCTGCAGGAGAGCGGCCCTGGCCTGGTG AAGCCTAGCGAGACACTGTCCCTGACCTGCGCCGTG AGCGGCTACAGCATCTCCAGCGGCTATTTCTGGGGA TGGATCAGACAGCCCCCTGGCAAGGGCCTGGAATGG ATCGGTTCTATCCTGCACTCCGGCGTGACATACTAT AACCCTAGCCTGAAGAGCAGGGTGACCATCTCCGTG GATACCAGCAAGAAT CAG T T CAGCC T GAAGC T CAGC AGCGTGACCGCCGCCGATACCGCTGTGTACTACTGC G C CAGAGACAG GAC CAC CGTCTCCATGATC GAG TAC

- 57 043303

TTCCAGCACTGGGGCCAAGGCACCCTGGTCACCGTG TCCTCCGCTAGCACAAAAGGACCAAGCGTGTTTCCA CTGGCACCTAGCAGCAAATCCACCAGCGGCGGAACA GCAGCCCTCGGGTGCCTGGTGAAGGATTACTTCCCT GAGCCAGTCACAGTGTCCTGGAACTCCGGAGCCCTG ACATCCGGCGTGCACACCTTCCCCGCTGTGCTGCAA TCCAGCGGACTGTATAGCCT CAGCTCCGTCGTGACA GTCCCTTCCAGCAGCCTGGGCACACAGACTTACATT T G CAAC G T GAAC СACAAAC C T T C CAACAC TAAG GTG GACAAAAAG GTG GAAC C CAAAT C C T G T GATAAGAC C CATACATGCCCACCTTGTCCCGCTCCTGAGCTGCTG GGGGGACCTTCCGTCTTTCTGTTTCCTCCAAAACCA AAAGACACAC T CAT GAT CAG C C G GAC C C C C GAAG T C ACCTGTGTGGTGGTGGACGTCAGCCACGAAGATCCA GAGGTCAAGTTCAATTGGTACGTGGATGGAGTGGAA GTC CACAAC G CAAAAAC CAAAC WITH TAGAGAAGAACAG TACAATAG CACATACAG GGTGGTGTCCGTCCT GAC A GTGCTCCACCAGGACTGGCTCAATGGCAAAGAGTAT AAGTGCAAGGTGAGCAACAAGGCCCTGCCTGCACCA AT T GAGAAAACAAT TAGCAAGGCAAAGGGGCAGCCA CGGGAACCCCAGGTGTATACCCTGCCCCCAAGCCGG GAT GAAC T GAC CAAAA AC CAG G T CAG CCT GACAT G C CTGGTGAAAGGGTTTTTACCCAAGCGATATTGCCGTC GAGTGGGAGAGCAACGGACAGCCAGAAAACAATTAC AAAACCACCCCACCTGTGCTGGACTCCGATGGGAGC TTTTTCCTG TACAG CAAG C T CACAG T G GACAAG TCC AGATGGCAACAGGGCAACGTGTTTTCCTGCTCCGTG ATG CAC GAG G C C C T C CACAAC CAC TATACACAAAAG TCCCTCTCCCTCAGCCCAGGAAAGTGA 49 NS IgGl aFXI- 18611 (QD (L105) SSGLY SLSSVVTVPSSSLGTQTYICNVNHKPSNTKV

- 58 043303

DKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTEMPERTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK 50 DNA encoding NS IgGl aFXI18611 (QI) (L105) xxx= CAG or CAA (Q) xxxGTCCAGCTGCAGGAGAGCGGCCCTGGACTCGTG AAGCCCTCCGAAACCCTGAGCCTCACATGCGCCGTC TCCGGATACAGCATCAGCAGCGGATACTTCTGGGGC TGGATCAGACAGCCCCCCGGCAAAGGCCTGGAGTGG ATCGGTTCTATTCTC CACAG C G G C G T GACATAC TAC AACCCCTCCCTGAAGAGCAGGGTGACCATCAGCGTG GACAC C T C CAAGAAC CAG TTTTCCCT CAAG C T GAG C AGCGTGACCGCCGCTGACACAGCCGTGTATTACTGC G C CAG G GACAG GAC CAC CGTGTCCCTGATT GAG TAC TTCCAGCATTGGGGCCAGGGCACACTGGTGACCGTC AGCAGCGCTAGCACAAAAGGACCAAGCGTGTTTCCA CTGGCACCTAGCAGCAAATCCACCAGCGGCGGAACA GCAGCCTCGGGTG CCTGGTGAAGGATTACTTCCCT GAGCCAGTCACAGTGTCCTGGAACTCCGGAGCCCTG ACATCCGGCGTGCACACCTTCCCCGCTGTGCTGCAA TCCAGCGGACTGTATAGCCTCAGCTCCGTCGTGACA GTCCCTTCCAGCAGCCTGGGCACACAGACTTACATT T G CAAC G T GAAC CACAAAC C T T C CAACAC TAAG GTG GACAAAAAG GTG GAAC C CAAAT C C T G T GATAAGAC C CATACATGCCCACCTTGTCCCGCTCCTGAGCTGCTG GGGGGACCTTCCGTCTTTCTGTTTCCTCCAAAACCA AAAGACACAC T CAT GAT CAG CCG GAC C C C C GAAG T C ACCTGTGTGGTGGTGGACGTCAGCCACGAAGATCCA GAGGTCAAGTTCAATTGGTACGTGGA TGGAGTGGAA GTC C AC AAC G CAAAAAC CAAAC C T AGAGAAGAAC AG TACAATAG CACATACAG GGTGGTGTCCGTCCT GACA GTGCTCCACCAGGACTGGCTCAATGGCAAAGAGTAT AAGTGCAAGGTGAGCAACAAGGCCCTGCCTGCACCA

- 59 043303

AT T GAGAAAACAAT TAGCAAGGCAAAGGGGCAGCCA CGGGAACCCCAGGTGTATACCCTGCCCCCAAGCCGG GAT GAAC T GAC CAAAAAC CAG G T CAG C C T GACAT G C CTGGTGAAAGGGTTTTTACCCAAGCGATATTGCCGTC GAGTGGGAGAGCAACGGACAGCCAGAAAACAATTAC AAAACCACCCCACCTGTGCTGGACTCCGATGGGAGC TTTTT CCTG TAGAG CAAG C T CACAG T G GACAAG T C C AGATGGCAACAGGGCAACGTGTTTTCCTGCTCCGTG AT G CAC GAG G C C C T C CACAAC CAC TATACACAAAAG TCCCTCTCCCTCAGCCCAGGAAAGTGA 51 NS IgGl aFXI- 18611 (El)(L105) EVOLOESGPGLVKPSETLSLTCAVSGYSISSGYFWG WIRQPPGKGLEWIGSILHSGVTYYNPSLKSRVTISV DT S KNQ FS LKL S SVTAADTAVYYCARDRTTVSLIEY FQHWGOGTLVTVSSASTKGPSVFPEAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVP SSSLGTQTYICNVNHKPSNTKV DKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTEMPERTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK 52 DNA encoding NS IgGl aFXI18611 (El)(L105) xxx=GAA or GAG (E) xxxGTCCAGCTGCAGGAGAGCGGCCCTGGACTCGTG AAGCCCTCCGAAACCCTGAGCCTCACATGCGCCGTC TCCGGATACAGCATCAGCAGCGGATACTTCTGGGGC TGGATCAGACAGCCCCCCGGCAAAGGCCTGGAGTGG ATCGGTTCTATTCTC CACAG C G G C G T GACATAC TAG AACCCCTCCCTGAAGAGCAGGGTGACCATCAGCGTG GACAC C T C CAAGAAC CAG TTTTCCCT CAAG C T GAG C AGCGTGACCGCCGCTGACACAGCCGTGTATTACTGC G CAG G GACAG GAC CAC CGTGTCCCTGATT GAG TAG TTCCAGCATTGGGGCCAGGGCACACTGGTGACCGTC AGCAGCGCTAGCACAAAAGGACCAAGCGTGTTTCCA CTGGCACCTAGCAGCAAATCCACCAGCGGCGGAACA

- 60 043303

GCAGCCCTCGGGTGCCTGGTGAAGGATTACTTCCCT GAGCCAGTCACAGTGTCCTGGAACTCCGGAGCCCTG ACATCCGGCGTGCACACCTTCCCCGCTGTGCTGCAA TCCAGCGGACTGTATAGCCTCAGCTCCGTCGTGACA GTCCCTTCCAGCAGCCTGGGCACACAGACTTACATT T G CAAC G T GAAC СACAAAC C T T C CAACAC TAAG GTG GA CAAAAAG GTG GAAC C CAAAT C C T G T GATAAGAC C CATACATGCCCACCTTGTCCCGCTCCTGAGCTGCTG GGGGGACCTTCCGTCTTTCTGTTTCCTCCAAAACCA AAAGACACAC T CAT GAT CAG CCG GAC C C C C GAAG T C ACCTGTGGTGGTGGACGTCAGCCACGAAGATCCA GAGGTCAAGTTCAATTGGTACGTGGATGGAGTGGA A GTC CACAAC G CAAAAAC CAAAC C TAGAGAAGAACAG TACAATAG CACATACAG GGTGGTGTCCGTCCT GACA GTGCTCCACCAGGACTGGCTCAATGGCAAAGAGTAT AAGTGCAAGGTGAGCAACAAGGCCCTGCCTGCACCA AT T GAGAAAACAAT TAGCAAGGCAAAGGGGCAGCCA CGGGAACCCCAGGTGTATACCCTGCCCCCAAGCCGG GAT GAAC T GAC CAAAAAC CAG G T CAG CCT GACAT G C CTGGTGAAAGGGTTTTACCAAGCGATATTGCCGTC GAGTGGGAGAGCAACGGACAGCCAGAAAACAATTAC AAAACCACCCCACCTGTGCTGGACTCCGATGGGAGC TTTTTCCTG TACAG CAAG C T CACAG T G GACAAG T C C AGATGGCAACAGGGCAACGTGTTTTCCTGCTCCGTG ATG CAC GAG G C C T C CACAAC CAC TATACACAAAAG TCCC TCTCCCTCAGCCCAGGAAAGTGA 53 HC IgGl aFXI18623p (IQ) QVQLQESGPGLVKPSQTLSLTСTVSGGSIYSGAYYW S WIRQHPGKGLEWIGSIHYSGLTYYNPSLKSRVTIS VDTSKNOFSLKLSSVTAADTAVYYCARDVDDSSGDE HYGMDVWGQGTTVTVSS ASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWESGALTSGVHTFPA VLQSSGLY SLSSVVTVPSSSLGTQTYLCEVEHKPSE TKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFEWYVD GVEVHNAKTKPREEQYESTYRVVSVLTVLHQDWLEG

- 61 043303

KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK 54 DNA encoding NS IgGl αΚΧΙ18623р (1Q) xxx= CAG or CAA (Q) xxxGTCCAGCTGCAGGAATCCGGACCCGGCCTGGTG AAGCCTAGCCAGACCCTGAGCCTGACCTGTACCGTG TCCGGCGGAAGCATCTATTCCGGCGCCTACTACTGG TCCTGGATTAGGCAGCACCCCGGCAAGGGCCTGGAA TGGATCGGCTCCATCCACTACAGCGGCCTGACCTAT TACAACCCCTCCCTGAAGTCCAGGGTGACCATCAGC GTC GACACAAG CAAGAAC CAG TTCTCCCT CAAG CTG AGCAGCGTGACCGCCGCCGACACCGCCGTGTATTAT T G C G C C AGAGAC GTG GAC GAC T C C T C C G GAGAC GAG CACTACGGCATGGACGTCTGGGGCCAGGGCACAACA GTGACAGTGAGCAGCGCTAGCACAAAAGGACCAAGC GTGTTTCCACTGGCACCTAGCAGCAAATCCACCAGC GGCGGAACAGCAGCCCTCG GGTGCCTGGTGAAGGAT TACTTCCCTGAGCCAGTCACAGTGTCCTGGAACTCC GGAGCCCTGACATCCGGCGTGCACACCTTCCCCGCT GTGCTGCAATCCAGCGGACTGTATAGCCTCAGCTCC GTCGTGACAGTCCCTTCCAGCAGCCTGGGCACACAG AC T TACAT T T G CAAC G T GAAC СACAAAC C T T C CAAC ACTAAGGTGGACAAAAGGTGGAACCCAAA TССTGT GATAAGACCCATACATGCCCACCTTGTCCCGCTCCT GAGCTGCTGGGGGGACCTTCCGTCTTTCTGTTTCCT C CAAAAC CAAAAGACACAC T CAT GAT CAG C G GAC C CCCGAAGTCACCTGTGTGGTGGTGGACGTCAGCCAC GAAGAT C CAGAG G T CAAG T T CAAT TGGTACGTGGAT G GAG T G GAAG T C CACAAC G CAAAAAC CAAA C C TAGA GAAGAACAGTACAATAGCACATACAGGGTGGTGTCC GTCCTGACAGTGCTCCACCAGGACTGGCTCAATGGC AAAGAGTATAAGTGCAAGGTGAGCAACAAGGCCCTG CCTGCACCAATTGAGAAAACAATTAGCAAGGCAAAG GGGCAGCCACGGGAACCCCAGGTGTATACCCTGCCC CCAAGCCGGGATGAACTGACCAAAAACCAGGTCAGC

- 62 043303

CTGACATGCCTGGTGAAAGGGTTTTTACCCAAGCGAT ATTGCCGTCGAGTGGGAGAGCAACGGACAGCCAGAA AACAAT TACAAAAC CAC C C CAC CTGTGCTG GAC T C C GATGGGAGCTTTTTCCTGTACAGCAAGCTCACAGTG GACAAGTCCAGATGGCAACAGGGCAACGTGTTTTCC TGCTCCGTGATGCACGAGGCCCTCCACAACCAT ACACAAAAG TCCCTCTCCCTCAGCCCAGGAAAGTGA 55 NS IgGl aFXI18623p (IE) EVOLOESGPGLVKPSOTLSLTCTVSGGSIYSGAYYW S WIRQHPGKGLEWIGSIHYSGLTYYNPSLKSRVTIS VDTSKNOFSLKLSSVTAADTAVYYCARDVDDSSGDE HYGMDVWGOGTTVTVSS ASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVP PSRDELTKNQVSLTCLVK GFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK 56 DNA encoding NS IgGl αΕΧΙ18623p (IE) xxx=GAA or GAG (E) xxxGTCCAGCTGCAGGAATCCGGACCCGGCCTGGTG AAGCCTAGCCAGACCCTGAGCCTGACCTGTACCGTG TCCGGCGGAAGCATCTATTCCGGCGCCTACTACTGG TCCTGGATTAGGCAGCACCCCGGCAAGGGCCTGGAA TGGATCGGCTCCATCCACTACAGCGGCCTGACCTAT TACAACCCCTCCCTGAAGTCCAGGGTGACCATCAGC GTC GACACAAG CAAGAAC CAG TTCTCCCT CAAG C T G AGCAGCGTGACCGCCGCCGACACCGCCGTGTATTAT T G C G C C AGAGAC GTG GAC GAC T C C T C G GAGAC GAG CACTACGGCATGGACGTCTGGGGCCAGGGCACAACA GTGACAGTGAGCAGCGCTAGCACAAAAGGACCAAGC GTGTTTCCACTGGCACCTAGCAGCAAATCCACCAGC GGCGGAACAGCAGCCCT CGGGTGCCTGGTGAAGGAT TACTTCCCTGAGCCAGTCACAGTGTCCTGGAACTCC GGAGCCCTGACATCCGGCGTGCACACCTTCCCCGCT

- 63 043303

GTGCTGCAATCCAGCGGACTGTATAGCCTCAGCTCC GTCGTGACAGTCCCTTCCAGCAGCCTGGGCACACAG ACTTACATTTG CAAC G T GAACCACAAACC T T CCAAC ACTAAGGTGGACAAAAAGGTGGAACCCAAATSCTGT GATAAGACCCATACATGCCCACCTTGTCCCGCTCCT GAGCTGCTGGGGGGACCTTCCGTCTTTCTGTTTCCT C CAAAAC CAAAAGA CACAC T CAT GAT CAG C C G GAC C CCCGAAGTCACCTGTGTGGTGGTGGACGTCAGCCAC GAAGAT C CAGAG G T CAAG T T CAAT TGGTACGTGGAT G GAG T G GAAG T C CACAAC G CAAAAAC CAAAC C TAGA GAAGAACAG TACAATAG CACATACAG GGTGGTGTCC GTCCTGACAGTGCTCCACCAGGACTGGCTCAATG GC AAAGAGTATAAGTGCAAGGTGAGCAACAAGGCCCTG CCTGCACCAATTGAGAAAACAATTAGCAAGGCAAAG GGGCAGCCACGGGAACCCCAGGTGTATACCCTGCCC CCAAGCCGGGATGAACTGACCAAAAACCAGGTCAGC CTGACATGCCTGGTGAAAGGGTTTTTACCCAAGCGAT ATTGCCGTCGAGTGGGAGAGCAACGGACAGCCAGAA AACAAT TACAAAAC CAC C C CAC CTGTGCTG GAC T C C GATGGGAGCTTTTTCCTGTACAGCAAGCTCACAGTG GACAAG TCCAGATGGCAACAGGGCAACGTGTTTTCC TGCTCCGTGATGCACGAGGCCCTCCACAACCACTAT ACACAAAAGTCCCTCTCCCTCAGCCCAGGAAAGTGA 57 HC-IgG4 aFXI18611p (S228P) (QI) (M105) (No terminal K) QVQLQESGPGLVKPSETLSLTCAVSGYSISSGYFWG WIRQP P GKGLEWIGSILHSGVTYYNPSLKSRVTISV DT S KNQ FS LKL S SVTAADTAVYYCARDRTTVSMIEY FQHWGQGTLVTVSS ASTKGPSVFPLAPCSRSTSEST AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLY T KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE

- 64 043303

ALHNHYTQKSLSLSSLG 58 DNA encoding HC-IgG4 aFXI- 18611r (S228P)(QI) (Ml 05) ; xxx= GAG or CAA (Q) (C-terminal K absent) xxxGTCCAGCTGCAGGAGAGCGGCCCTGGCCTGGTG AAGCCTAGCGAGACACTGTCCCTGACCTGCGCCGTG AGCGGCTACAGCATCTCCAGCGGCTATTTCTGGGGA TGGATCAGACAGCCCCCTGGCAAGGGCCTGGAATGG ATCGGTTCTATCCTGCACTCCGGCGTGACATACTAT AACCCTAGCCTGAAGAGCAGGGTGACCATCTCCGTG GATACCAGCAAGAAT CAG T T CAGCC T GAAGC T CAGC AGCGTGACCGCCGCCGATACCGCTGTGTACTACTGC G C CAGAGACAG GAG GAG CGTCTCCATGATC GAG TAG TTCCAGCACTGGGGCCAAGGCACCCTGGTCACCGTG TCCTCCGCCTCCACCAAGGGCCCTAGCGTGTTTCCT CTGGCCCCCTGCTCCAGATCCACAAGCGAGAGCACC GCTGCCCTGGGCTGTCTGGTCAAGGACT ACTTCCCC GAGCCCGTGACAGTGTCCTGGAACAGCGGCGCCCTG ACAAGCGGCGTCCATACATTCCCCGCCGTGCTGCAG TCCAGCGGACTGTATAGCCTGAGCTCCGTGGTGACC GTGCCTTCCAGCAGCCTGGGAACCAAGACATATACC TGCAACGTGGAGCATAAGCCCAGCAACACAAAAGTC GACAAGAGGGTGGAGAGCAAGTACGGACCCCCTTGT CCCCCTTGTC CTGCTCCCGAGTTCCTCGGCGGACCT AGCGTGTTCCTGTTTCCTCCCAAGCCCAAGGATACC CTGATGATCAGCAGGACCCCTGAGGTCACCTGCGTG GTGGTCGACGTGTCCCAGGAGGACCCTGAGGTCCAG TTTAACTGGTACGTGGACGGAGTGGAGGTGCACAAC G C CAAGAC CAAG C C CAGAGAG GAG CAG T T CAAT T C C ACCTACAGGGTGGTGAGCGTCCTGACCGTGCTGCAC CAGGACTGGCTGAATGGAAAGGAGTACAAATGCAAG GTCTCCAACAAGGGCCTCCCTAGCAGCATCGAGAAG ACCATCTCCAAGGCCAAGGGCCAGCCTAGGGAGCCC CAGGTGTACACCCTGCCTCCTAGCCAGGAGGAAATG ACCAAGAACCAGGTGTCCCTGACATGCCTGGTGAAG GGCTTCTATCCTAGCGACATCGCC GTGGAGTGGGAG AGCAATGGCCAGCCCGAGAATAACTACAAGACСACC CCCCCTGTGCTCGATAGCGACGGCAGCTTCTTTCTG

- 65 043303

TACAGCAGGCTGACCGTGGACAAGAGCAGGTGGCAA GAGGGCAACGTGTTTAGCTGCTCCGTCATGCACGAG GCCCTGCATAACCACTACACCCAAAAATCCCTGTCC CTGTCCCTGGGGC 59 HC-IgG4 aFXI18611p (S228P) (El) (M105) (No terminal K) EVOLOESGPGLVKPSETLSLTCAVSGYSISSGYFWG WIRQP P GKGLEWIGSILHSGVTYYNPSLKSRVTISV DT S KNQ FS LKL S SVTAADTAVYYCARDRTTVSMIEY FQHWGOGTLVTVSS ASTKGPSVFPLAPCSRSTSEST AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVT VPSSSLGTKTYTCNVDHKPSNTKV DKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDT LMLSRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHN AKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKGLPSSLEKTLSKAKGQPREPQVYTLPPSQEEM TKNQV SLTCLVKGFYPSDTAVEWESNGQPENNYKTT PPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLG 60 DNA encoding HC-IgG4 aFXI18611р S228P); (El) (M105) xxx=GAA or GAG (E) (C-terminal K missing) xxxGTCCAGCTGCAGGAGAGCGGCCCTGGCCTGGTG AAGCCTAGCGAGACACTGTCCCTGACCTGCGCCGTG AGCGGCTACAGCATCTCCAGCGGCTATTTCTGGGGA TGGATCAGACAGCCCCCTGGCAAGGGCCTGGAATGG ATCGGTTCTATCCTGCACTCCGGCGTGACATACTAT AACCCTAGCCTGAAGAGCAGGGTGACCATCTCCGTG GATACCAGCAAGAAT CAG T T CAGCC T GAAGC T CAGC AGCGTGACCGCCGCCGATACCGCTGTGTACTACTGC G C CAGAGACAG GAC CAC CGTCTCCATGATC GAG TAC TTCCAGCACTGGGGCCAAGGCACCCTGGTCACCGTG TCCTCCGCCTCCACCAAGGGCCCTAGCGTGTTTCCT CTGGCCCCCTGCTCCAGATCCACAAGCGAGAGCACC GCTGCCCTGGGCTGTCTGGTCAAGGACT ACTTCCCC GAGCCCGTGACAGTGTCCTGGAACAGCGGCGCCCTG ACAAGCGGCGTCCATACATTCCCCGCCGTGCTGCAG TCCAGCGGACTGTATAGCCTGAGCTCCGTGGTGACC GTGCCTTCCAGCAGCCTGGGAACCAAGACATATACC TGCAACGTGGACCATAAGCCCAGCAACACAAAAGTC

- 66 043303

GACAAGAGGGTGGAGAGCAAGTACGGACCCCCTTGT CCCCCTTGTCCTGCTCCCGAGTTCCTCGGCGGACCT AGCGTGTTCCTGTTTCCTCCCAAGCCCAAGGATACC CTGATGATCAGCAGGACCCCTGAGGTCACCTGCGTG GTGGTCGACGTGTCCCAGGAGGACCCTGAGGTCCAG TTTAACTGGTACGTGGACGGAGTGGAGGTGCACAAC G С CAAGAC CAAG С С CAGAGAG GAG CAG T T CAAT TCC ACCTACAGGGTGGTGAGCGTCCTGACCGTGCTGCAC CAGGACTGGCTGAATGGAAAGGAGTACAAATGCAAG GTCTCCAACAAGGGCCTCCCTAGCAGCATCGAGAAG ACCATCTCCAAGGCCAAGGGCCAGCCTAGGGAGCCC CAGGTGTACACCCTGCCTCCTAGCCAGGAGGAAATG ACCAAGAACCAGGTGTCCCTGACATGCCTGGTGAAG GGCTTCTATCCTAGCGACATCGCCGTGGAGTGGGAG AGCAATGGСCAGССCGAGAATAACTACAAGACСACC CCCCCTGTGCTCGATAGCGACGGCAGCTTCTTTCTG TACAGCAGGCTGACCGTGGACAAGAGCAGGTGGCAA GAGGGCAACGTGTTTAGCTGCTCCGTCATGCACGAG GCCCTGCATAACCACTACACCCAAAAATCCCTGTCC CTGTCCCTGGGC 61 HC-IgG4 aFXI18611 S228P) (QI) (L105) (No terminal K) OVOLQESGPGLVKPSETLSLTCAVSGYSISSGYFWG WIRQPPGKGLEWIGSILHSGVTYYNPSLKSRVTISV DT S KNQ FS LKL S SVTAADTAVYYCARDRTTVSLIEY FQHWGQGTLVTVSS ASTKGPSVFPLAPCSRSTSEST AALGCLVKDYFPEPVTVSWESGALTSGVHTFPAVLQ SSGLYSLS SVVTVPSSSLGTKTYTCEVDHKPSETKV DKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDT LMLSRTPEVTCVVVDVSQEDPEVQFEWYVDGVEVHE AKTKPREEQFNSTYRVVSVLTVLHQDWLEGKEYKCK VSEKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEM TKEQV SLTCLVKGFYPSDLAVEWESEGQPEEEYKTT PPVLDSDGSFFLYSRLTVDKSRWQEGEVFSCSVMHE ALHNHYTQKSLSLSLG 62 DNA encoding HC-IgG4 aFXI- xxxGTCCAGCTGCAGGAGAGCGGCCCTGGACTCGTG AAGCCCTCCGAAACCCTGAGCCTCACATGCGCCGTC

- 67 043303

18611 (S228P); (QD (L105) xxx=CAG or CAA (Q) (Terminal K absent) TCCGGATACAGCATCAGCAGCGGATACTTCTGGGGC TGGATCAGACAGCCCCCCGGCAAAGGCCTGGAGTGG ATCGGTTCTATTCTC CACAG C G G C G T GACATAC TAC AACCCCTCCCTGAAGAGCAGGGTGACCATCAGCGTG GACAC C T C CAAGAAC CAG TTTTCCCT CAAG C T GAG C AGCGTGACCGCCGCTGACACAGCCGTGTATTACTGC G C CAG G GACAG GAC CAC CGTGTCCCTGATT GAG TAC TTCCAGCATTGGGGCCAGGGCACACTGGTGACCGTC AGCAGCGCCAGCACCAAGGGCCCTTCCGTCTTCCCT CTGGCCCCTTGCAGCAGAAGCACCTCCGAGTCCACA GCCGCCCTGGGATGCCTCGTGAAGGATTACTTCCCC GAGCCCGTCACAGTCTCCTGGAACTCCGGCGCTCTG ACCAGCGGAG TGCACACCTTCCCCGCCGTGCTGCAA AGCAGCGGCCTGTACAGCCTGTCCAGCGTGGTCACC GTGCCTTCCTCCAGCCTGGGCACCAAGACCTACACA T G CAAC GTG GAC CACAAG C C T T C CAACAC CAAG GTG GACAAGAGAGTGGAAAGCAAGTACGGCCCCCCCTGC CCCCCTTGTCCTGCCCCCGAGTTTCTGGGAGGACCC TCCGTGTTCCTCTTTCCTCCCAAGCCTAAGGACACC CTGATGATCTCCAGGACCCCCGAAGTGACCTGCGTG GTCGTGGACGTGTCCCAGGAGGACCCTGAGGTGCAG TTTAACTGGTACGTGGACGGCGTGGAGGTGCACAAC GCCAAGACCAAGC CCAGGGAGGAGCAGTTCAATAGC ACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCAC CAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAA GTCAGCAACAAGGGCCTGCCCTCCTCCATCGAGAAG ACCATTAGCAAGGCCAAGGGCCAGCCTAGGGAGCCT CAGGTGTACACCCTGCCCCCCAGCCAGGAGGAGATG ACCAAGAACCAGGTGTCCCTGACCTGCCTGGTCAAG GGATT TTACCCCAGCGACATCGCTGTGGAATGGGAG AGCAATGGCCAGCCCGAGAACAACTACAAGACСACC CCTCCCGTGCTCGATTCCGACGGCAGCTTTTTCCTG TACAGCAGGCTGACCGTGGATAAGAGCAGGTGGCAG GAAGGCAACGTGTTCTCCTGTTCCGTGATGCATGAG G C C C T G CACAAC CAC TACACACAGAAGAG C C T G T C C

- 68 043303

CTGTCCCTGGGGC 63 HC-IgG4 aFXI18611 (S228P) (El) (L105) (No terminal K) EVOLOESGPGLVKPSETLSLTCAVSGYSISSGYFWG WIRQPPGKGLEWIGSILHSGVTYYNPSLKSRVTISV DTSKNOFSLKLSSVTAADTAVYYCARDRTTVSLIEY FQHWGOGTLVTVSS ASTKGPSVFPLAPCSRSTSEST AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGT KTYTCNVDHKPSNTKV DKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDT LMLSRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHN AKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKGLPSSTEKTLSKAKGQPREPQVYTLPPSQEEM TKNQVSLTCLVKGF YPSDLAVEWESNGQPENNYKTT PPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLG 64 DNA encoding HC-IgG4 aFXI18611 (S228P) (QI) (L105) xxx=GAA or GAG (E) (C-terminal K missing) xxxGTCCAGCTGCAGGAGAGCGGCCCTGGACTCGTG AAGCCCTCCGAAACCCTGAGCCTCACATGCGCCGTC TCCGGATACAGCATCAGCAGCGGATACTTCTGGGGC TGGATCAGACAGCCCCCCGGCAAAGGCCTGGAGTGG ATCGGTTCTATTCTC GAGAG C G G C G T GACATAC TAG AACCCCTCCCTGAAGAGCAGGGTGACCATCAGCGTG GACAC C T C CAAGAAC CAG TTTTCCCT CAAG C T GAG C AGCGTGACCGCCGCTGACACAGCCGTGTATTACTGC G C CAG G GACAG GAC CAC CGTGTCCCTGATT GAG TAG TTCCAGCATTGGGGCCAGGGCACACTGGTGACCGTC AGCAGCGCCAGCACCAAGGGCCCTTCCGTCTTCCCT CTGGCCCCTTGCAGCAGAAGCACCTCCGAGTCCACA GCCGCCCTGGG ATGCCTCGTGAAGGATTACTTCCCC GAGCCCGTCACAGTCTCCTGGAACTCCGGCGCTCTG ACCAGCGGAGTGCACACCTTCCCCGCCGTGCTGCAA AGCAGCGGCCTGTACAGCCTGTCCAGCGTGGTCACC GTGCCTTCCTCCAGCCTGGGCACCAAGACCTACACA T G CAAC G T G GAC CACAAG C C T T C CAACAC CAAG G T G GACAAGAGAGTGGAAAGCAAGTACGGCCCCCCCTGC CCCCCTTGTCCTGCCCCCGAGTTTCTGGGAGGACCC TCCGTGTTCCTCTTTCCTCCCAAGCCTAAGGACACC

- 69 043303

CTGATGATCTCCAGGACCCCCGAAGTGACCTGCGTG GTCGTGGACGTGTCCCAGGAGGACCCTGAGGTGCAG TTTAACTGGTACGTGGACGGCGTGGAGGTGCACAAC GCCAAGACCAAGCCCAGGGAGGAGCAGTTCAATAGC ACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCAC CAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAA GTCAGCAACAAGGGCCTGCC CTCCTCCATCGAGAAG ACCATTAGCAAGGCCAAGGGCCAGCCTAGGGAGCCT CAG GTG TACAC CCTGCCCCC CAG C CAG GAG GAGAT G ACCAAGAACCAGGTGTCCCTGACCTGCCTGGTCAAG GGATTTTACCCCAGCGACATCGCTGTGGAATGGGAG AG CAAT GGC CAG CCC GAGAACAAC TACAAGAC CAC C CCTCCCGTGCTCGATTCCGACGGC AGCTTTTTCCTG TACAGCAGGCTGACCGTGGATAAGAGCAGGTGGCAG GAAGGCAACGTGTTCTCCTGTTCCGTGATGCATGAG G C C C T G CACAAC CAC TACACACAGAAGAG C C T G T C C CTGTCCCTGGGC 65 HC-IgG4 αΕΧΙ- 18623p (S228P( (QD(C- terminal K absent) QVQLQESGPGLVKPSQTLSLTCTVSGGSIYSGAYYW SWIRQHPGKGLEWIGSIHYSGLTYYNPSLKSRVTIS VDTSKNQFSLKLSSVTAADTAVYYCARDVDDSSGDE HYGMDVWGQGT TVTVS S ASTKGPSVFPLAPCSRSTS ESTAALGCLVKDYFPEPVTVSWKSGALTSGVHTFPA VLQSSG LYSLSSVVTVPSSSLGTKTYTCNVDHKPSN TKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQ EEMT KNQVSLTCLVKGFYPSDLAVEWESNGQPENNY KTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV MHEALHNHYTQKSLSLSLG 66 DNA encoding HC-IgG4 aFXI- 18623r (S228P( (QI) xxx= CAG or CAA (Q) (C- xxxGTCCAGCTGCAGGAATCCGGACCCGGCCTGGTG AAGCCTAGCCAGACCCTGAGCCTGACCTGTACCGTG TCCGGCGGAAGCATCTATTCCGGCGCCTACTACTGG TCCTGGATTAGGCAGCACCCCGGCAAGGGCCTGGAA TGGATCGGCTCCATCCACTACAGCGGCCTGACCTAT

- 70 043303

terminal K absent) TACAACCCCTCCCTGAAGTCCAGGGTGACCATCAGC GTC GACACAAG CAAGAAC CAG TTCTCCCT CAAG C T G AGCAGCGTGACCGCCGCCGACACCGCCGTGTATTAT T G C G C C AGAGAC GTG GAC GAC T C C T C G GAGAC GAG CACTACGGCATGGACGTCTGGGGCCAGGGCACAACA GTGACAGTGAGCAGCGCCACCAAAG GACCCTCC GTCTTCCCTCTGGCCCCTTGCTCCAGGAGCACAAGC GAAAGCACAGCCGCCCTGGGCTGCCTGGTGAAGGAC TACTTTCCCGAGCCCGTGACCGTGAGCTGGAATAGC GGAGCCCTCACCTCCGGAGTCCACACATTTCCCGCC GTCCTGCAGAGCAGCGGCCTGTACTCCCTGAGCTCC GTGGTGACCGTGCCTTCCTCCAGCCTGGGCACCAAG AC WITH TACAC WITH T G CAAC GTG GAC CACAAG C C TAG CAAT ACCAAGGTGGACAAGAGGGTGGAATCCAAGTACGGC CCCCCTTGCCCTCCTTGTCCTGCCCCCGAATTTCTG GGCGGCCCTTCCGTGTTCCTGTTCCCTCCCAAGCCC AAG GATAC C C T GAT GAT CAG CAG GAC C C C T GAG GTG ACCTGTGGTGGTGGACGTGAGCCAGGAGGACCCC GAGGTGCAGTTCAACTGGTACGTGGATGGCGTGGAA GTGCACAATGCCAAGACAAAGCCCAGGGAGGAGCAG TTCAATAGCACCTACAGGGTGGTCAGCGTGCTCACA GTGCTGCACCAGGACTGGCTGAACGGAAAGGAGTAC AAGTGC AAAGTGTCCAACAAGGGCCTGCCCTCCTCC ATCGAAAAGACCATCTCCAAGGCCAAAGGCCAGCCC AGGGAGCCCCAAGTGTATACCCTCCCCCTAGCCAG GAG GAAAT GAC C AAAAAC C AG G T C T C C C T GAC C T G T CTGGTGAAGGGCTTCTATCCCAGCGACATCGCTGTG GAGTGGGAGAGCAACGGCCAACCCGAGAACAACTAT AAGACCACACCCC CCGTCCTGGACTCCGATGGCTCC TTCTTCCTGTACAGCAGGCTGACCGTCGACAAGTCC AGGTGGCAGGAAGGAAACGTGTTCTCCTGTAGCGTC ATG CAC GAG G C C C T G CACAAC CAC TATAC C CAGAAG TCCCTGTCCCTGAGCCTGGGC 67 HC-IgG4 αΕΧΙ- 18623r (S228P( EVQLQESGPGLVKPSQTLSLTCTVSGGSIYSGAYYW SWIRQHPGKGLEWIGSIHYSGLTYYNPSLKSRVTIS

- 71 043303

(El) (terminal K missing) VDTSKNQFSLKLSSVTAADTAVYYCARDVDDSSGDE HYGMDVWGQGT TVTVS S ASTKGPSVFPLAPCSRSTS ESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSN TKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKP KDTLMIS RTPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQ EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV MHEALHN HYTQKSLSLSLG 68 DNA encoding HC-IgG4 aFXI18623р (S228P( (El) xxx=GAA or GAG (E) (Terminal K missing) xxxGTCCAGCTGCAGGAATCCGGACCCGGCCTGGTG AAGCCTAGCCAGACCCTGAGCCTGACCTGTACCGTG TCCGGCGGAAGCATCTATTCCGGCGCCTACTACTGG TCCTGGATTAGGCAGCACCCCGGCAAGGGCCTGGAA TGGATCGGCTCCATCCACTACAGCGGCCTGACCTAT TACAACCCCTCCCTGAAGTCCAGGGTGACCATCAGC GTC GACACAAG CAAGAAC CAG TTCTCCCT CAAG C T G AGCAGCGTGACCGCCGCCGACACCGCCGTGTATTAT T G C G C C AGAGAC GTG GAC GAC T C C T C G GAGAC GAG CACTACGGCATGGACGTCTGGGGCCAGGGCACAACA GTGACAGTGAGCAGCGCCAGCACCAAAGGACCCTCC GTCTTCCCTCTGGCCCCTTGCTCCAGGAGCACAAGC GAAAGCACAGCCGCCCTG C CCCCTTGCCCTCCTTGTCCTGCCCCCGAATTTCTG GGCGGCCCTTCCGTGTTCCTGTTCCCTCCCAAGCCC AAG GATAC C C T GAT GAT CAG CAG GAC C C C T GAG GTG ACCTGTGGTGGTGGACGTGAGCCAGGAGGACCCC GAGGTGCAGTTCAACTGGTACGTGGATGGCGTGGAA

- 72 043303

GTGCACAATGCCAAGACAAAGCCCAGGGAGGAGCAG TTCAATAGCACCTACAGGGTGGTCAGCGTGCTCACA GTGCTGCACCAGGACTGGCTGAACGGAAAGGAGTAC AAGTGCAAAGTGTCCAACAAGGGCCTGCCCTCCTCC ATCGAAAAGACCATCTCCAAGGCCAAAGGCCAGCCC AGGGAGCCCCAAGTGTATACCCTCCCCCCTAGCCAG GAGGAAATGACCAAAAASS AGGТСТСССТGACCТGТ CTGGTGAAGGGCTTCTATCCCAGCGACATCGCTGTG GAGTGGGAGAGCAACGGCCAACCCGAGAACAACTAT AAGACCACACCCCCCGTCCTGGACTCCGATGGCTCC TTCTTCCTGTACAGCAGGCTGACCGTCGACAAGTCC AGGTGGCAGGAAGGAAACGTGTTCTCCTGTAGCGTC AT G CAC GAG G C C C T G CACAAS CAC TATAS WITH CAGAAG TCCCTGTCCCTGAGCCTGGGC 69 NS IgGl αΕΧΙ18611p (QI) (M105) (C-terminal K missing) OVOLOESGPGLVKPSETLSLTCAVSGYSISSGYFWG WIRQP P GKGLEWIGSILHSGVTYYNPSLKSRVTISV DT S KNQ FS LKL S SVTAADTAVYYCARDRTTVSMIEY FQHWGQGTLVTVSSASTKGPSVFPYAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPG 70 DNA encoding NS IgGl αΕΧΙ18611р (Q1) (Ml 05) xxx= CAG or CAA (Q) (C- terminal K absent) xxxGTCCAGCTGCAGGAGAGCGGCCCTGGCCTGGTG AAGCCTAGCGAGACACTGTCCCTGACCTGCGCCGTG AGCGGCTACAGCATCTCCAGCGGCTATTTCTGGGGA TGGATCAGACAGCCCCCTGGCAAGGGCCTGGAATGG ATCGGTTCTATCCTGCACTCCGGCGTGACATACTAT AACCCTAGCCTGAAGAGCAGGGTGACCATCTCCGTG GATACCAGCAAGAAT CAG T T CAGCC T GAAGC T CAGC AGCGTGACCGCCGCCGATACCGCTGTGTACTACTGC

- 73 043303

G C CAGAGACAG GAC CAC CGTCTCCATGATC GAG TAC TTCCAGCACTGGGGCCAAGGCACCCTGGTCACCGTG TCCTCCGCTAGCACAAAAGGACCAAGCGTGTTTCCA CTGGCACCTAGCAGCAAATCCACCAGCGGCGGAACA GCAGCCCTCGGGTGCCTGGTGAAGGATTACTTCCCT GAGCCAGTCACAGTGTCCTGGAACTCCGGAGCCCTG ACATCCGGCGTGCA CACCTTCCCCGCTGTGCTGCAA TCCAGCGGACTGTATAGCCTCAGCTCCGTCGTGACA GTCCCTTCCAGCAGCCTGGGCACACAGACTTACATT T G CAAC G T GAAC СACAAAC C T T C CAACAC TAAG GTG GACAAAAAG GTG GAAC C CAAAT C C T G T GATAAGAC C CATACATGCCCACCTTGTCCCGCTCCTGAGCTGCTG GGGGGACCTTCCG TCTTTCTGTTTCCTCCAAAACCA AAAGACACAC T CAT GAT CAG CCG GAC C C C C GAAG T C ACCTGTGTGGTGGTGGACGTCAGCCACGAAGATCCA GAGGTCAAGTTCAATTGGTACGTGGATGGAGTGGAA GTC CACAAC G CAAAAAC CAAAC C TAGAGAAGAACAG TACAATAG CACATACAG GGTGGTGTCCGTCCT GACA GTGCTCCACCAGGACTGGCTCAATGGCAAAGAGTAT AAGTGCAAGGTGAGCAACAAGGCCCTGCCT GCACCA AT T GAGAAAACAAT TAGCAAGGCAAAGGGGCAGCCA CGGGAACCCCAGGTGTATACCCTGCCCCCAAGCCGG GAT GAAC T GAC CAAAAAC CAG G T CAG CCT GACAT G C CTGGTGAAAGGGTTTTTACCCAAGCGATATTGCCGTC GAGTGGGAGAGCAACGGACAGCCAGAAAACAATTAC AAAACCACCCCACCTGTGCTGGACTCCGATGGGAGC TTTTT CCTG TACAG CAAG C T CACAG T G GACAAG TCC AGATGGCAACAGGGCAACGTGTTTTCCTGCTCCGTG ATG CAC GAG G C C C T C CACAAC CAC TATACACAAAAG TCCCTCTCCCTCAGCCCAGGA 71 NS IgGl aFXI18611p (El) (M105) (C- terminal K absent) EVQLQESGPGLVKPSETLSLTCAVSGYSISSGYFWG WIRQPPGKGLEWIGSILHSGVTYYNPSLKSRVTISV DTSKNOFSLKLSSVTAADTAVYYCARDRTTVSMIEY FGBWGOGYLVYVSSASTKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ

- 74 043303

SSGLYSLSSVVTVPSSSLGTQTYLCNVNHKPSNTKV DKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTK NQVSLTCLVKGFYPSDLAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPG 72 DNA encoding NS IgGl αΚΧΙ18611р (Q1) (Ml 05) xxx=GAA or GAG (E) (C- terminal K absent) xxxGTCCAGCTGCAGGAGAGCGGCCCTGGCCTGGTG AAGCCTAGCGAGACACTGTCCCTGACCTGCGCCGTG AGCGGCTACAGCATCTCCAGCGGCTATTTCTGGGGA TGGATCAGACAGCCCCCTGGCAAGGGCCTGGAATGG ATCGGTTCTATCCTGCACTCCGGCGTGACATACTAT AACCCTAGCCTGAAGAGCAGGGTGACCATCTCCGTG GATACCAGCAAGAAT CAG T T CAGCC T GAAGC T CAGC AGCGTGACCGCCGCCGATACCGCTGTGTACTACTGC G C CAGAGACAG GAC CAC CGTCTCCATGATC GAG TAC TTCCAGCACTGGGGCCAAGGCACCCTGGTCACCGTG TCCTCCGCTAGCACAAAAGGACCAAGCGTGTTTCCA CTGGCACCTAGCAGCAAATCCACCAGCGGCGGAACA GCAGCCTCGGGTGCCTGGTGAAGGATTACT TCCCT GAGCCAGTCACAGTGTCCTGGAACTCCGGAGCCCTG ACATCCGGCGTGCACACCTTCCCCGCTGTGCTGCAA TCCAGCGGACTGTATAGCCTCAGCTCCGTCGTGACA GTCCCTTCCAGCAGCCTGGGCACACAGACTTACATT T G CAAC G T GAAC CACAAAC C T T C CAACAC TAAG GTG GACAAAAAG GTG GAAC C CAAAT C C T G T GATAAGAC C CATACATGCCCACCTTGTCCCGCTCCTGAGCTGCTG GGGGGACCTTCCGTCTTTCTGTTTCCTCCAAAACCA AAAGACACAC T CAT GAT CAG C C G GAC C C C GAAG T C ACCTGTGTGGTGGTGGACGTCAGCCACGAAGATCCA GAGGTCAAGTTCAATTGGTACGTGGATGGAGTGGAA GTC CACAAC G CAAAAAC CAAAC C TAGAGAAAGAACAG TACAATAG CACATACAG GGTGGTGTCCGTCCT GACA GTGCTCCACCAGGACTGGCTCAATGGCAAAGAGTAT

- 75 043303

AAGTGCAAGGTGAGCAACAAGGCCCTGCCTGCACCA AT T GAGAAAACAAT TAGCAAGGCAAAGGGGCAGCCA CGGGAACCCCAGGTGTATACCCTGCCCCCAAGCCGG GAT GAAC T GAC CAAAAAC CAG G T CAG CCT GACAT G C CTGGTGAAAGGGTTTTACCCAAGCGATATTGCCGTC GAGTGGGAGAGCAACGGACAGCCAGAAAACAATTAC AAAA CCACCCCACCTGTGCTGGACTCCGATGGGAGC TTTTTCCTG TACAG CAAG C T CACAG T G GACAAG T C C AGATGGCAACAGGGCAACGTGTTTTCCTGCTCCGTG ATG CAC GAG G C C C T C CACAAC CAC TATACACAAAAG TCCCTCTCCCTCAGCCCAGGA 73 HC IgGl aFXI18611 (Ql) (L105) (C- terminal K absent) QVQLQESGPGLVKPSETLSLTCAVSGYSISSGYFWG WIRQPPGKGLEWIGSILHSGVTYYNPSLKSRVTISV DT S KNQ FS LKL S SVTAADTAVYYCARDRTTVSLIEY FQHWGOGYLVYVSSASTKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWESGALTSGVHTFPAVLQ SSGLYS LSSVVTVPSSSLGTQTYLCEVEHKPSETKV DKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMLSRTPEVTCVVVDVSHEDPEVKFEWYVDGVE VHEAKTKPREEQYESTYRVVSVLTVLHQDWLEGKEY KCKVSEKALPAPLEKTLSKAKGQPREPQVYTLPPSR DELTKEQVSLTCLV KGFYPSDLAVEWESEGQPEEEY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGEVFSCSV MHEALHEHYTQKSLSLSPG 74 DNA encoding NS IgGl aFXI18611 (Ql) (L105) xxx= CAG or CAA (Q) (C-terminal K absent) xxxGTCCAGCTGCAGGAGAGCGGCCCTGGACTCGTG AAGCCCTCCGAAACCCTGAGCCTCACATGCGCCGTC TCCGGATACAGCATCAGCAGCGGATACTTCTGGGGC TGGATCAGACAGCCCCCCGGCAAAGGCCTGGAGTGG ATCGGTTCTATTCTC CACAG C G G C G T GACATAC TAC AACCCCTCCCTGAAGAGCAGGGTGACCATCAGCGTG GACAC C T C CAAGAAC CAG TTTTCCCT CAAG C T GAG C AGCGTGACCGCCGCTGACACAGCCGTGTATTACTGC G C CAG G GACAG GAC CAC CGTGTCCCTGATT GAG TAC TTCCAGCATTGGGGCCAGGGCACACTGGTGACCGTC AGCAGCGCTAGCACAAAAGGACCAAGCGTGTTTCCA

- 76 043303

CTGGCACCTAGCAGCAAATCCACCAGCGGCGGAACA GCAGCCCTCGGGTGCCTGGTGAAGGATTACTTCCCT GAGCCAGTCACAGTGTCCTGGAACTCCGGAGCCCTG ACATCCGGCGTGCACACCTTCCCCGCTGTGCTGCAA TCCAGCGGACTGTATAGCCTCAGCTCCGTCGTGACA GTCCCTTCCAGCAGCCTGGGCACACAGACTTACATT T G CAAC G T GAAC CACAAAC C T T C CAACAC TAAG GTG GACAAAAAG GTG GAAC C CAAAT C C T G T GATAAGAC C CATACATGCCCACCTTGTCCCGCTCCTGAGCTGCTG GGGGGACCTTCCGTCTTTCTGTTTCCTCCAAAACCA AAAGACACAC T CAT GAT CAG C C G GAC C C C C GAAG T C ACCTGTGTGGTGGTGGACGTCAGCCACGAAGATCCA GAGGTCAAGTTCAATTGGTACGTGGATGGAGTGGAA GTC CACAAC G CAAAAAC CAAAC WITH TAGAGAAGAACAG TACAATAG CACATACAG GGTGGTGTCCGTCCT GACA GTGCTCCACCAGGACTGGCTCAATGGCAAAGAGTAT AAGTGCAAGGTGAGCAACAAGGCCCTGCCTGCACCA ATTGAGAAAACAATTAGCAAGGCAAAGGGGCAGCCA CGGGAACCCCAGGTGTATACCCTGCCCCCAAGCCGG GAT GAAC T GAC CAAAAAC CAG G T CAG CCT GACAT G C CTGGTGAAAGGGTTTTTACCCAAGCGATATTGCCGTC GAGTGGGAGAGCAACGGACAGCCAGAAAACAATTAC AAAACCACCCCACCTGTGCTGGACTCCGATGGGAGC TTTTTCCTG TACAG CAAG C T CACAG T G GACAAG TCC AGATGGCAACAGGGCAACGTGTTTTCCTGCTCCGTG ATG CAC GAG G C C C T C CACAAC CAC TATACACA AAAG TCCCTCTCCCTCAGCCCAGGA 75 NS IgGl aFXI18611 (El) (L105) (C- terminal K absent) EVQLQESGPGLVKPSETLSLTCAVSGYSISSGYFWG WIRQP P GKGLEWIGSILHSGVTYYNPSLKSRVTISV DT S KNQ FS LKL S SVTAADTAVYYCARDRTTVSLIEY FQHWGOGTLVTVSSASTKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMTSRTPEVTCVVVDVSHEDPEVKFNWYVDGVE

- 77 043303

VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPG 76 DNA encoding NS IgGl aFXI18611 (El)(L105) xxx=GAA or GAG (E) (C-terminal K missing) xxxGTCCAGCTGCAGGAGAGCGGCCCTGGACTCGTG AAGCCCTCCGAAACCCTGAGCCTCACATGCGCCGTC TCCGGATACAGCATCAGCAGCGGATACTTCTGGGGC TGGATCAGACAGCCCCCCGGCAAAGGCCTGGAGTGG ATCGGTTCTATTCTC CACAG C G G C G T GACATAC TAC AACCCCTCCCTGAAGAGCAGGGTGACCATCAGCGTG GACAC C T C C AAGAACCAGTTTTCCCT CAAGС T GAGC AGCGTGACCGCCGCTGACACAGCCGTGTATTACTGC G C CAG G GACAG GAC CAC CGTGTCCCTGATT GAG TAC TTCCAGCATTGGGGCCAGGGCACACTGGTGACCGTC AGCAGCGCTAGCACAAAAGGACCAAGCGTGTTTCCA CTGGCACCTAGCAGCAAATCCACCAGCGGCGGAACA GCAGCCTCGGGTGCC TGGTGAAGGATTACTTCCCT GAGCCAGTCACAGTGTCCTGGAACTCCGGAGCCCTG ACATCCGGCGTGCACACCTTCCCCGCTGTGCTGCAA TCCAGCGGACTGTATAGCCTCAGCTCCGTCGTGACA GTCCCTTCCAGCAGCCTGGGCACACAGACTTACATT T G CAACGT GAACCACAAACC T T CCAACACTAAGGTG GACAAAAAG GTG GAAC C CAAAT SS T GT GATAAGACC CATACATGCCCACCTTGTCCCGCTCCTGAGCTGCTG GGGGGACCTTCCGTCTTTCTGTTTCCTCCAAAACCA AAAGACACACT CAT GAT CAGCCGGACCC CCGAAGT C ACCTGTGTGGTGGTGGACGTCAGCCACGAAGATCCA GAGGTCAAGTTCAATTGGTACGTGGATGGAGTGGAA GTC CAC AAC G CAAAAAC C AAAC C T AGAGAAGAAC AG TACAATAG CACATACAG GGTGGTGTCCGTCCT GAGA GTGCTCCACCAGGACTGGCTCAATGGCAAAGAGTAT AAGTGCAAGGTGAGCAACAAGGCCCTGCCTGCACCA ATTGAGAAAACAATTAGCAAGGCAAAGGGGCAGCCA CGGGAACCCCAGGTGTATACCCTGCCCCCAAGCCGG

- 78 043303

GAT GAAC T GAC CAAAAAC CAG G T CAG C S T GACAT G C CTGGTGAAAGGGTTTTACCAAGCGATATTGCCGTC GAGTGGGAGAGCAACGGACAGCCAGAAAACAATTAC AAAACCACCCCACCTGTGCTGGACTCCGATGGGAGC TTTTTCCTG TACAG CAAG S T CACAG T G GACAAG TCC AGATGGCAACAGGGCAACGTGTTTTCCTG CTCCGTG AT G CAC GAG G C S S T S CASAASSAS TATACACAAAAG TCCCTCTCCCTCAGCCCAGGA 77 HC IgGl aFXI18623p (IQ) (C-terminal K missing) OVOLOESGPGLVKPSOTLSLTCTVSGGSIYSGAYYW SWIRQHPGKGLEWIGSIHYSGLTYYNPSLKSRVTIS VDTSKNOFSLKLSSVTAADTAVYYCARDVDDSSGDE HYGMDVWGOGTTVTVSS ASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSN TKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPG 78 DNA encoding NS IgGl aFXI18623p (1Q) xxx= CAG or CAA (Q) (Terminal K missing) xxxGTCCAGCTGCAGGAATCCGGACCCGGCCTGGTG AAGCCTAGCCAGACCCTGAGCCTGACCTGTACCGTG TCCGGCGGAAGCATCTATTCCGGCGCCTACTACTGG TCCTGGATTAGGCAGCACCCCGGCAAGGGCCTGGAA TGGATCGGCTCCATCCACTACAGCGGCCTGACCTAT TACAACCCCTCCCTGAAGTCCAGGGTGACCATCAGC GTC GACACAAG CAAGAAC CAG TTCTCCCT CAAG CTG AGCAGCGTGACCGCCGCCGACACCGCCGTGTATTAT T G C G C C AGAGAC GTG GAC GAC T C C T C C G GAGAC GAG CACTACGGCATGGACGTCTGGGGCCAGGGCACAACA GTGACAGTGAGCAGCGCTAGCACAAAAGGACCAAGC GTGTTTCCACTGGCACCTAGCAGCAAATCCACCAGC GGCGGAACAGCAGCCCTCG GGTGCCTGGTGAAGGAT TACTTCCCTGAGCCAGTCACAGTGTCCTGGAACTCC

- 79 043303

GGAGCCCTGACATCCGGCGTGCACACCTTCCCCGCT GTGCTGCAATCCAGCGGACTGTATAGCCTCAGCTCC GTCGTGACAGTCCCTTCCAGCAGCCTGGGCACACAG AC T TACAT TTG CAAC G T GAAC СACAAAC C T T C CAAC ACTAAGGTGGACAAAAAGGTGGAACCCAAATCCTGT GATAAGACCCATACATGCCCACCTTGTCCCGCTCCT GAGCTGCTGG GGGGACCTTCCGTCTTTCTGTTTCCT C CAAAAC CAAAAGACACAC T CAT GAT CAG CCG GAC C CCCGAAGTCACCTGTGTGGTGGTGGACGTCAGCCAC GAAGATСCAGAGGT CAAGT T CAAT TGGTACGTGGAT G GAG T G GAAG T C CACAAC G CAAAAAC CAAAC C TAGA GAAGAACAG TACAATAG CACATACAG GGTGGTGTCC GTCCTGACAGTGCTCCACCAGGACTGGCTCAATGGC C AC C C CAC CTGTGCTG GAC T C C GATGGGAGCTTTTTCCTGTACAGCAAGCTCACAGTG GACAAGTCCAGATGGCAACAGGGCAACGTGTTTTCC TGCTCCGTGATGCACGAGGCCCTCCACAACCACTAT ACACAAAAGTCCCTCTCCCTCAGCCCAGGA 79 NS IgGl aFXI18623p (IE) (C-terminal K missing) EVOLQESGPGLVKPSOTLSLTCTVSGGSIYSGAYYW SWIRQHPGKGLEWIGSIHYSGLTYYNPSLKSRVTIS VDTSKNQFSLKLSSVTAADTAVYYCARDVDDSSGDE HYGMDVWGOGTTVTVSS ASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSN TKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSRDELTKNQVSLTC LVKGFYPSDIAVEWESNGQPE

- 80 043303

NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPG 80 DNA encoding NS IgGl αΕΧΙ18623р (IE) xxx=GAA or GAG (E) (C-terminal K is missing) xxxGTCCAGCTGCAGGAATCCGGACCCGGCCTGGTG AAGCCTAGCCAGACCCTGAGCCTGACCTGTACCGTG TCCGGCGGAAGCATCTATTCCGGCGCCTACTACTGG TCCTGGATTAGGCAGCACCCCGGCAAGGGCCTGGAA TGGATCGGCTCCATCCACTACAGCGGCCTGACCTAT TACAACCCCTCCCTGAAGTCCAGGGTGACCATCAGC G T S GACACAAG CAAGA AC CAG TTTSSSST CAAG C T G AGCAGCGTGACCGCCGCCGACACCGCCGTGTATTAT T G C G C C AGAGAC G T G GAC GAC T S S T S C G GAGAC GAG CACTACGGCATGGACGTCTGGGGCCAGGGCACAACA GTGACAGTGAGCAGCGCTAGCACAAAAGGACCAAGC GTGTTTCCACTGGCACCTAGCAGCAAATCCACCAGC GGCGGAACAGC AGCCCTCGGGTGCCTGGTGAAGGAT TACTTCCCTGAGCCAGTCACAGTGTCCTGGAACTCC GGAGCCCTGACATCCGGCGTGCACACCTTCCCCGCT GTGCTGCAATCCAGCGGACTGTATAGCCTCAGCTCC G T C G T GACAG T S S S T T S CAG CAG S S T G G G CACACAG AS T TASAT T T G CAAC G T GAAC CACAAAAS S T T S CAAS ASTAAGGTGGACAAAAAGGTGGAACSSAAATSSGT GATAAGACCCATACATGCCCACCTTGTCCCGCTCCT GAGCTGCTGGGGGGACCTTCCGTCTTTCTGTTTCCT C CAAAAC CAAAAGAC ACAC T CAT GAT CAG C C G GAC C CCCGAAGTCACCTGTGTGGTGGTGGACGTCAGCCAC GAAGAT C CAGAG G T CAAG T T CAAT TGGTACGTGGAT G GAG T G GAAG T C CACAAC G CAAAAAC CAAAC S TAGA GAAGAACAG TACAATAG CACATACAG GGTGGTGTCC GTCCTGACAGTGCTCCACCAGGACTGGCTCAATGGC AAAGAGTATAAGTGCAAGGTGAGCAACAAGGCCCTG CCTGCACCAATТGAGAAAACAATTA GCAAGGCAAAG GGGCAGCCACGGGAACCCCAGGTGTATACCCTGCCC CCAAGCCGGGATGAACTGACCAAAAACCAGGTCAGC CTGACATGCCTGGTGAAAGGGTTTTTACCCAAGCGAT ATTGCCGTCGAGTGGGAGAGCAACGGACAGCCAGAA

- 81 043303

81 82 83 84 85 Human FXI Epitope A Epitope B Light chain kappa antibodies to RSV NS IgG4 (S228P) to RSV AACAAT TASAAAAS CAC C C CAC CTGTGCTG GAC TCC GATGGGAGCTTTTTCCTGTACAGCAAGCTCACAGTG GACAAGTCCAGATGGCAACAGGGCAACGTGTTTTCC TGCTCCGTGATGCACGAGGCCCTCCACAACCATAT ACACAAAAGTCCCTCTCCCTCAGCCCAGGA ECVTQLLKDTCFEGGDITTVFTPSAKYCQWCTYHP RCLLFTFTAESPSEDPTRWFTCVLKDSVTETLPRVN RTAAIS GYS FKQC S HQISACNKDIYVDLDMKGIΝΥΝ SSVAKSAQECQERCTDDVHCHFFTYATRQFPSLEHR NICLLKHTQTGTPTRITKLDKWSGFSLKSCALSNL ACIRDIFPN TVFADSNIDSVMAPDAFVCGRICTHHP GCLFFTFFSQEWPKESQRNLCLLKTSESGLPSTRIK KSKALSGFSLQSCRHSIPVFCHSSFYHDTDFLGEEL DIVAAKSHEACQKLCTNAVRCQFFTYTPAQASCNEG KGKCYLKLSSNGSPTKILHGRGGISGYTLRLCKMDN ECTTKIKPRIVGGTASVRGEWPWQVTLHTTSPT QRH LCGGSIIGNQWILTAAHCFYGVESPKILRVYSGILN QSEIKEDTSFFGVQEIIIHDQYKMAESGYDIALLKL ETTVNYTDSQRPICLPSKGDRNVIYTDCWVTGWGYR KLRDKIQNTLQKAKIPLVTNEECQKRYRGHKITNKM ICAGYREGGKflACKGDSGGPLSCKHNEVWHLVGITS WGEGCAQRERPGVYTNWEYVDWILEKTQAV DIFPNTVF PSTRIKKSKALSG MAPVQLLGLLVLFLPAMRCDIQMTQSPSTLSASVGD RVTITCKCQLSVGYMHWYQQKPGKAPKLLIYDTSKL ASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCFQ GSGYPFTFGGGTKLEIKRTVAAPSVFIFPPSDEQLK SGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES VTEQDSKDS TYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGEC MAVVQLLGLLVLFLPAMRCQVTLRESGPALVKPTQT LTLTCTFSGFSLSTSGMSVGWIRQPPGKALEWLADI WWDDKKDYNPSLKSRLTISKDTSKNQWLKVTNMDP ADTATYYCARSMITNWYFDVWGAGTTVTVSSASTKG PSVFFLAPGSRS TSESTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPE FLGGPSVFLFPPKPKDTLMLSRTPEVTCVVVDVSQE DPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKGLPSSTEKTTSKAKG QPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDL AVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVD KSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Constant regions are shown in italics. The underlined amino acid sequences represent CDRs.

Although the present invention has been described herein with reference to illustrative embodiments, it should be understood that the invention is not limited thereto. Those of ordinary skill in the art and having access to the description herein will understand further modifications and embodiments within its scope. Therefore, the present invention is limited only by the claims appended hereto.

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Claims (33)

CLAIM 1. An antibody or antigen-binding fragment that binds to the apple 3 domain of coagulation factor XI (FXI), containing: (a) a heavy chain complementarity determining region (HC-CDR) 1 having the amino acid sequence shown in SEQ ID NO: 8, HC-CDR 2 having the amino acid sequence shown in SEQ ID NO: 9, and HC-CDR 3, having the amino acid sequence shown in SEQ ID NO: 10; and (b) a light chain complementarity determining region (LC-CDR) 1 having the amino acid sequence shown in SEQ ID NO: 11, LC-CDR 2 having the amino acid sequence shown in SEQ ID NO: 12, and LC-CDR 3 having the amino acid sequence shown in SEQ ID NO: 13, wherein the antibody or antigen binding fragment is for the treatment of a thromboembolic disorder or disease. 2. The antibody or antigen binding fragment of claim 1, wherein the antibody or antigen binding fragment comprises a heavy chain variable domain having the amino acid sequence shown in SEQ ID NO: 28 or 29 and a light chain variable domain having the amino acid sequence shown in SEQ ID NO: 28 or 29 NO: 30. 3. The antibody or antigen binding fragment of claim 1 or 2, wherein the antibody comprises a heavy chain constant domain comprising the amino acid sequence shown in SEQ ID NO: 16, 17, 18 or 19. 4. An antibody or antigen binding fragment according to any one of claims 1 to 3, wherein the antibody comprises a light chain constant domain comprising the amino acid sequence shown in SEQ ID NO: 20. 5. An antibody or antigen-binding fragment according to claim 1, where the antibody or antibody fragment contains: (a) a heavy chain variable domain having the amino acid sequence shown in SEQ ID NO: 28 and a light chain variable domain having the amino acid sequence shown in SEQ ID NO: 30; or (b) a heavy chain variable domain having the amino acid sequence shown in SEQ ID NO: 29 and a light chain variable domain having the amino acid sequence shown in SEQ ID NO: 30. 6. The antibody or antigen binding fragment of claim 5, wherein the antibody further comprises a heavy chain constant domain comprising the amino acid sequence shown in SEQ ID NO: 16, 17, 18 or 19. 7. The antibody or antigen binding fragment of claim 5, wherein the antibody further comprises a light chain constant domain comprising the amino acid sequence shown in SEQ ID NO: 20. 8. The antibody or antigen binding fragment according to claims 1 to 7, wherein the antibody or antigen binding fragment binds to the apple 3 domain of coagulation factor XI (FXI) and inhibits activation of FXI and/or factor XIa-mediated activation of factor IX. 9. An antibody or antigen-binding fragment according to claim 1, where the antibody contains: (a) a heavy chain with a constant domain and a variable domain, wherein the variable domain contains a heavy chain complementarity determining region (HC-CDR) 1 with the amino acid sequence shown in SEQ ID NO: 8, HC-CDR 2 with the amino acid sequence shown in SEQ ID NO: 9, and HC-CDR 3 with the amino acid sequence shown in SEQ ID NO: 10; and (b) a light chain with a constant domain and a variable domain, wherein the variable domain comprises a light chain complementarity determining region (LC-CDR) 1 with the amino acid sequence shown in SEQ ID NO: 11, LC-CDR 2 with the amino acid sequence shown in SEQ ID NO: 12, and LC-CDR 3 with the amino acid sequence shown in SEQ ID NO: 13. 10. The antibody or antigen binding fragment of claim 9, wherein the antibody comprises a heavy chain constant domain comprising the amino acid sequence shown in SEQ ID NO: 16, 17, 18 or 19. 11. The antibody or antigen binding fragment of claim 9, wherein the antibody comprises a light chain constant domain comprising the amino acid sequence shown in SEQ ID NO: 20. 12. The antibody or antigen binding fragment of claim 1, wherein the antibody comprises a heavy chain with the amino acid sequence shown in SEQ ID NO: 41, 43, 53, 55, 65, 67, 77 or 79; and a light chain with the amino acid sequence shown in SEQ ID NO: 31, wherein the antibody or antigen binding fragment binds to the apple 3 domain of coagulation factor XI (FXI) and inhibits activation of FXI and/or factor XIa-mediated activation of factor IX. 13. Isolated nucleic acid molecule encoding the light chain variable domain - 83 043303 any of the antibodies or antigen-binding fragments according to claims 1-12. 14. An isolated nucleic acid molecule encoding the heavy chain variable domain of any of the antibodies or antigen-binding fragments according to claims 1-12. 15. An isolated nucleic acid molecule encoding a light chain variable domain and a heavy chain variable domain of any of the antibodies or antigen binding fragments of claims 1 to 12. 16. A composition for the treatment of a thromboembolic disorder or disease, comprising an antibody or antigen-binding fragment according to any one of claims 1 to 12 and a pharmaceutically acceptable carrier or diluent. 17. Use of an antibody according to any one of claims 1 to 12 for the preparation of a medicament for the treatment of a thromboembolic disorder or disease. 18. A method for producing an antibody or antigen-binding fragment containing: (i) a heavy chain variable domain comprising a heavy chain complementarity determining region (HC-CDR) 1 with the amino acid sequence shown in SEQ ID NO: 8, HCCDR 2 with the amino acid sequence shown in SEQ ID NO: 9, and HC-CDR 3 with the amino acid sequence shown in SEQ ID NO: 10; and (ii) a light chain variable domain comprising a light chain complementarity determining region (LC-CDR) 1 with the amino acid sequence shown in SEQ ID NO: 11, LC-CDR 2 with the amino acid sequence shown in SEQ ID NO: 12, and LC-CDR 3 with the amino acid sequence shown in SEQ ID NO: 13, a method comprising creating a host cell containing a nucleic acid molecule encoding a heavy chain and a nucleic acid molecule encoding a light chain; and culturing the host cell under conditions and for a period of time sufficient to produce an antibody or antigen-binding fragment. 19. The method of claim 18, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 28 or 29 and the light chain variable region contains the amino acid sequence of SEQ ID NO: 30. 20. The method according to claim 18, where the antibody contains a heavy chain constant domain of the IgG1, IgG2, IgG3 or IgG4 isotype. 21. The method according to claim 18, where the antibody contains a heavy chain constant domain of the IgG4 isotype. 22. The method of claim 18, wherein the antibody comprises a heavy chain constant domain comprising the amino acid sequence shown in SEQ ID NO: 16, 17, 18 or 19. 23. The method of claim 18, wherein the light chain comprises human kappa light chain or human lambda light chain. 24. The method of claim 18, wherein the antibody comprises a light chain constant domain comprising the amino acid sequence shown in SEQ ID NO: 20. 25. The method of claim 18, wherein the host cell is a Chinese hamster ovary cell or a human embryonic kidney cell 293. 26. The method of claim 18, wherein the host cell is a yeast or filamentous fungus cell. 27. An antibody or antigen binding fragment that binds to the apple 3 domain of coagulation factor XI (FXI), comprising a heavy chain variable domain (VH) comprising the amino acid sequence shown in SEQ ID NO: 29 and a light chain variable domain ( _VL ). containing the amino acid sequence shown in SEQ ID NO: 30. 28. The antibody or antigen binding fragment of claim 27, wherein the antibody further comprises a heavy chain constant domain having the amino acid sequence shown in SEQ ID NO: 16 or 17, and a light chain constant domain having the amino acid sequence shown in SEQ ID NO: 20. 29. The antibody or antigen binding fragment of claim 25, wherein the antibody further comprises an IgG1 isotype heavy chain constant domain that does not contain N-glycosylation of asparagine at position 297. 30. A composition for the treatment of a thromboembolic disorder or disease, comprising the antibody or antigen binding fragment of claim 27 and a pharmaceutically acceptable carrier or diluent. 31. An antibody that binds to the apple 3 domain of coagulation factor XI (FXI) comprising a heavy chain having the amino acid sequence shown in SEQ ID NO: 43 and a light chain having the amino acid sequence shown in SEQ ID NO: 31. 32. A composition for the treatment of a thromboembolic disorder or disease comprising an antibody that binds to the apple 3 domain of coagulation factor XI (FXI), wherein the antibody comprises a heavy chain having the amino acid sequence shown in SEQ ID NO: 43 and a light chain having the amino acid sequence the sequence shown in SEQ ID NO: 31, and a pharmaceutically acceptable carrier or diluent. 33. An antibody that binds to the apple 3 domain of coagulation factor XI (FXI), comprising a heavy chain having the amino acid sequence shown in SEQ ID NO: 67 and a light chain -