Bispecific antibodies and methods of use

Abstract

The present invention relates to bispecific antibodies comprising a first antigen-binding domain that specifically binds to TfR and a second and optionally a third antigen-binding domain that specifically binds to PD1. The invention further relates to methods of producing these molecules, methods of their use, pharmaceutical compositions thereof, and their use as medicaments for the treatment of cancer, acute and chronic infections, and graft-versus-host disease.

Description

[Technical field]

[0001] The present invention relates to a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second and optionally a third antigen-binding domain that specifically binds to PD1. The present invention further relates to immunoconjugates comprising the bispecific antibody, and methods of using the bispecific antibody or immunoconjugate. [Background technology]

[0002] The programmed death 1 protein (PD1 or CD279) is an inhibitory member of the CD28 family of cell surface receptors, which also includes CD28, CTLA-4, ICOS and BTLA, and binds to PD-L1 and PD-L2 (Greenwald RJ et al. Annu Rev Immunol. (2005) 23:515-48; Freeman GJ et al. J Exp Med. (2000) 192:1027-34; Latchman Y. et al. Nat Immunol. (2001) 2:261-8), forming the so-called PD1 / PD-L1 axis. PD1 is expressed on activated B cells, T cells, and myeloid cells (Agata et al, supra; Okazaki et al (2002) Curr. Opin. Immunol. 14:391779-82; Bennett et al. (2003) J Immunol 170:711-8). The PD1 gene is a 55 kDa type I transmembrane protein that is part of the Ig gene superfamily (Agata et al. (1996) Int Immunol 8:765-72). PD1 contains a membrane-proximal immunoreceptor tyrosine-based inhibitory motif (ITIM) and a membrane-distal tyrosine-based switch motif (ITSM) (Thomas, ML (1995) J Exp Med 181:1:1953-6; Vivier, E. and Daeron, M. (1997) Immunol Today 18:286-91). PD1 is structurally similar to CTLA-4, but lacks the MYPPPY motif (SEQ ID NO: 71) that is important for B7-1 and B7-2 binding. Two ligands for PD1, PD-L1 (CD274) and PD-L2 (CD273), have been identified and shown to downregulate T cell activation upon binding to PD1 (Freeman et al. (2000) J Exp Med 192:1027-34; Latchman et al (2001) Nat Immunol 2:261-8; Carter et al. (2002) Eur J Immunol 32:634-43). Both PD-L1 and PD-L2 are B7 homologs that bind to PD1 but not other CD28 family members.One ligand for PD1, PD-L1, is abundant in various human cancers (Dong et al (2002) Nat. Med 8:787-9). Targeting the PD1 / PD-L1 immunological checkpoint with monoclonal antibodies and small molecule drugs has become a major focus in immuno-oncology.

[0003] In addition to its role as an inhibitory member of the CD28 family, PD1 has been found to play a role in autoimmune encephalomyelitis, systemic lupus erythematosus, graft-versus-host disease (GVHD), type I diabetes, and rheumatoid arthritis (Salama et al. (2003) J Exp Med 198:71-78; Prokunina and Alarcon-Riquelme (2004) Hum MoI Genet 13:R143; Nielsen et al. (2004) Lupus 13:510). In mouse B cell tumor lines, the ITSM of PD1 inhibits Ca mediated by the B cell receptor. 2+ It has been shown that IL-1 is essential for blocking the flux of IL-1 and tyrosine phosphorylation of downstream effector molecules (Okazaki et al. (2001) PNAS 98:13866-71).

[0004] Various patent applications disclose methods of enhancing immune responses using the production of anti-PD1 antibodies and / or agents that interfere with PD-L1 binding and / or PD1 signaling, including anti-PD1 antibodies, including: U.S. Patent No. 2003 / 0039653, U.S. Patent No. 2004 / 0213795, U.S. Patent No. 2006 / 0110383, U.S. Patent No. 2007 / 0065427, U.S. Patent No. 2007 / 0122378, U.S. Patent No. 2012 / 237522, WO 2004 / 072286, WO 2006 / 121168, WO 2006 / 133396, WO 2007 / 005874, WO 2007 / 005875, WO 2007 / 005711, WO 2007 / 005912, WO 2007 / 005913, WO 2007 / 005914, WO 2007 / 005915, WO 2007 / 005916, WO 2007 / 005917, WO 2007 / 005919 ... International Publication No. 2008 / 083174, International Publication No. 2008 / 156712, International Publication No. 2009 / 024531, International Publication No. 2009 / 014708, International Publication No. 2009 / 114335, International Publication No. 2010 / 027828, International Publication No. 2010 / 027423, International Publication No. 2010 / 036959, International Publication No. WO 2010 / 029435, WO 2010 / 029434, WO 2010 / 063011, WO 2010 / 089411, WO 2011 / 066342, WO 2011 / 110604, WO 2011 / 110621 and WO 2012 / 145493.

[0005] The transferrin receptor (TfR) is a membrane receptor involved in iron transport into cells by binding to the iron-transferrin complex and internalizing it by receptor-mediated endocytosis. TfR is an attractive target for therapeutic approaches of intracellular delivery due to its rapid internalization and recycling rates. However, in vivo delivery is mostly inefficient and nonspecific due to the vast TfR expression throughout the body.

[0006] The effects of PD1 antibodies described in the art rely on blocking the interaction between PD-L1 and PD1 by binding to PD1. Because even the highest affinity antibody binding of anti-PD1 antibodies to PD1 is non-covalent and therefore transient, there is a need to develop new compounds that target PD1 with improved efficacy and longer-lasting effects than known anti-PD1 antibodies. Summary of the Invention

[0007] In one embodiment, the present invention provides a novel bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second and optionally a third antigen-binding domain that specifically binds to a molecule of the PD1 / PD-L1 axis. In one embodiment, the PD1 / PD-L1 axis molecule is selected from the group consisting of PD1, PD-L1 and PD-L2. In one particular embodiment, the PD1 / PD-L1 axis molecule is PD1 or PD-L1. In one particular embodiment, the PD1 / PD-L1 axis molecule is PD1. The anti-TfR anti-PD1 bispecific antibodies of the present invention have particularly advantageous properties, such as functionally optimized binding affinity, increased biological activity, specific targeting of certain T cells, and high targeting efficiency.

[0008] In another embodiment, the bispecific antibody binds to the TfR and PD1 receptors on the surface of cells that express and display TfR and PD1 on their surface. In a preferred embodiment, the binding of the antibody to TfR and PD1 is simultaneous. Binding of the bispecific antibody to TfR and PD1 on the cell surface preferably results in depletion of PD1 from the surface of the cell expressing TfR and PD1, by internalization into the cell of the complex formed by the bispecific antibody with TfR and PD1. This preferably results in depletion of PD1 from the cell surface, together with TfR and the bound bispecific antibody. The present invention is based at least in part on the discovery that the anti-PD1 anti-TfR bispecific antibody of the present invention has the advantageous effect of inhibiting the interaction between PD1 and PD-L1 by removing PD1 from the cell surface, which is more effective and / or more durable than the inhibition achievable by mere binding of an anti-PD1 blocking antibody that does not result in internalization of PD1 and depletion of PD1 from the cell surface.

[0009] In one embodiment, the present invention provides a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR, a second antigen-binding domain that specifically binds to PD1, and a third antigen-binding domain that specifically binds to PD1. Thus, the bispecific antibody has one antigen-binding domain specific to TfR and two antigen-binding domains specific to PD1. Such molecules with two binding domains for a first target and one binding domain for a second target are also referred to as 2+1 format or 2+1 format antibodies. In one embodiment, these molecules are based on IgG class Fab fragments and, optionally, IgG class Fc regions, which can be covalently linked to each other in different conformations to result in different 2+1 format antibodies. Examples of different 2+1 formats with different conformations of the antigen-binding domains are shown in Figures 1 to 4. Additional conformations have been described in the art (Brinkmann and Kontermann (2017) MAbs 9(2):182-212; Kontermann and Brinkmann (2015) Drug Discov Today 20(7):838-47; Bacac M et al. (2018) Clin Cancer Res. 24(19):4785-4797; Rius Ruiz et al. (2018) Sci Transl Med 10(461):eaat1445; Seckinger et al. (2017) Cancer Cell. 31(3):396-410; Bacac et al. (2016) Oncoimmunology. 5(8):e1203498; Bacac et al. (2016) Clin Cancer Res. 22(13):3286-97; Weber et al. (2018) Cell Rep.22(1):149-162;Niewoehner et al.(2014)Neuron.81(1):49-60).

[0010] Surprisingly, it has been found that such anti-TfR anti-PD1 2+1 format antibodies, i.e. bispecific antibodies with a 2:1 stoichiometry of the binding domains targeting anti-PD1 and anti-TfR, respectively, or in other words bispecific antibodies comprising a first antigen-binding domain that specifically binds to TfR and a second and a third antigen-binding domain that specifically binds to PD1, exhibit improved biological activity over monospecific bivalent PD1 antibodies and result in better inhibition of the interaction between PD1 and PD-L1.

[0011] In one embodiment of a bispecific antibody comprising a first antigen-binding domain specifically binding to TfR and a second and optionally a third antigen-binding domain specifically binding to PD1, the first antigen-binding domain, the second antigen-binding domain, and / or the third antigen-binding domain, if present, are Fab fragments. In a further embodiment, the invention relates to a bispecific antibody comprising a first antigen-binding domain specifically binding to TfR and a second and optionally a third antigen-binding domain specifically binding to PD1, the bispecific antibody comprising an Fc domain composed of a first and a second subunit. In a particular embodiment, one or more of the Fab fragments comprised in the bispecific antibody are fused to the Fc domain. In another embodiment, the Fab fragment is fused to the Fc domain via a peptidic linker. In an additional embodiment, the Fc domain is an IgG Fc domain, in particular an IgG1 Fc domain or an IgG4 Fc domain. In a particular embodiment, the heavy chain of the bispecific antibody is of the gamma type (IgG), in particular of the gamma type. In another specific embodiment, the light chains of the bispecific antibody are of the kappa (κ) and / or lambda (λ) subtype, based on the amino acid sequence of their constant domains.

[0012] In one embodiment, the present invention provides a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second and optionally a third antigen-binding domain that specifically binds to PD1, the bispecific antibody comprising an Fc domain, a first Fab fragment comprising an antigen-binding domain that specifically binds to TfR, and a second and optionally a third Fab fragment comprising an antigen-binding domain that specifically binds to PD1. In a particular embodiment, the bispecific antibody comprises an Fc domain, a first Fab fragment comprising an antigen-binding domain that specifically binds to TfR, and a second and optionally a third Fab fragment comprising an antigen-binding domain that specifically binds to PD1, the Fab fragments being fused to the Fc domain. In one embodiment, the bispecific antibody comprises exactly one (monovalent) antigen-binding domain that specifically binds to TfR and exactly two (monovalent) antigen-binding domains that specifically bind to PD1. In particular, the Fc domain is an IgG Fc domain, more particularly an IgG1 Fc domain or an IgG4 Fc domain. In one particular embodiment, the heavy chain of the bispecific antibody is of the gamma type (IgG), in particular of the gamma 1 (IgG1) subtype. In another particular embodiment, the light chain of the bispecific antibody is of the kappa (κ) and / or lambda (λ) subtype, based on the amino acid sequence of its constant domain. In one embodiment, the bispecific antibody does not comprise a J chain. In another embodiment, the bispecific antibody does not comprise a hybrid IgA / IgG antibody sequence and / or a hybrid IgM / IgG antibody sequence. In a further embodiment, the bispecific antibody is essentially in monomeric form, i.e. does not form dimeric or multimeric (e.g. pentameric) structures comprising a plurality of bispecific antibodies of the invention. In a particular embodiment, at least 90%, more particularly at least 95%, preferably at least 98%, and even more preferably at least 99% of the antibody is in monomeric form.

[0013] In one aspect, the invention relates to a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second and optionally a third antigen-binding domain that specifically binds to PD1, wherein the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor, in particular an Fcγ receptor. In particular, the Fc domain is of the human IgG1 subclass with the amino acid mutations L234A, L235A and P329G (numbering according to the Kabat EU index).

[0014] In another aspect, the invention relates to a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second and optionally a third antigen-binding domain that specifically binds to PD1, wherein the Fc domain comprises a modification that promotes association of the first and second subunits of the Fc domain.In one aspect, the invention relates to a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second and optionally a third antigen-binding domain that specifically binds to PD1, wherein the first subunit of the Fc domain comprises a knob and the second subunit of the Fc domain comprises a hole according to the knobs into holes method. In a particular embodiment, the bispecific antibody is one in which the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W (numbering according to Kabat EU index) and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S and Y407V (numbering according to Kabat EU index).

[0015] In a further aspect, the invention provides a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second and optionally a third antigen-binding domain that specifically binds to PD1, wherein the first antigen-binding domain, the second antigen-binding domain and, if present, the third antigen-binding domain are each Fab fragments, and in one or two of the Fab fragments: a) the variable domains VL and VH are swapped with each other such that the VH domain is part of a light chain and the VL domain is part of a heavy chain, or b) A bispecific antibody is provided in which the constant domains CL and CH1 are replaced by each other such that the CH1 domain is part of the light chain and the CL domain is part of the heavy chain.

[0016] In a preferred embodiment, the variable domains VL and VH are replaced with each other such that the VH domain is part of the light chain and the VL domain is part of the heavy chain. In a particular embodiment, the bispecific antibody is one in which either the variable domains VL and VH or the constant domains CL and CH1 are replaced with each other in a Fab fragment comprising an antigen-binding domain that specifically binds to PD1. In a particularly preferred embodiment, the variable domains VL and VH are replaced with each other in an antigen-binding domain that specifically binds to PD1. In one embodiment, the bispecific antibody comprises exactly one (monovalent) antigen-binding domain that specifically binds to TfR and exactly two (monovalent) antigen-binding domains that specifically bind to PD1.

[0017] In an additional aspect, the invention relates to a bispecific antibody comprising a first antigen-binding domain which specifically binds to TfR and a second and optionally a third antigen-binding domain which specifically binds to PD1, wherein the first antigen-binding domain, the second antigen-binding domain and, if present, the third antigen-binding domain are each Fab fragments, and wherein in one or two of the Fab fragments in the constant domain CL the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat EU index) and in the constant domain CH1 the amino acids at positions 147 and 213 are independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index). In a particular embodiment, the invention relates to a bispecific antibody comprising a first antigen-binding domain which specifically binds to TfR and a second and optionally a third antigen-binding domain which specifically binds to PD1, wherein in the Fab fragment comprising the antigen-binding domain which specifically binds to TfR, in the constant domain CL, the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat EU index) and in the constant domain CH1, the amino acids at positions 147 and 213 are independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index).

[0018] In a particular embodiment, the invention provides a bispecific antibody comprising a first antigen-binding domain which specifically binds TfR and a second and optionally a third antigen-binding domain which specifically binds PD1, wherein the second Fab fragment comprising the antigen-binding domain which specifically binds PD1 and, if present, the third Fab fragment, in the constant domain CL, have the amino acid at position 124 independently substituted by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat EU index) and the amino acids at positions 147 and 213 independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index).

[0019] In one embodiment, a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second antigen-binding domain that specifically binds to PD1 comprises: a) a first light chain and a first heavy chain of an antibody that specifically binds to a first antigen; b) A bivalent antibody that specifically binds to a second antigen, comprising a second light chain and a second heavy chain of an antibody, wherein the variable domains VL and VH of the second light chain and the second heavy chain are replaced by each other, and the constant domains CL and CH1 of the second light chain and the second heavy chain are replaced by each other.

[0020] The two subunits of the antibody of a) do not contain the modifications reported in b), and the heavy and light chains of a) are isolated chains. In the two subunits of the antibody of b), in the light chain the variable light domain VL is replaced by the variable heavy domain VH of said antibody and the constant light domain CL is replaced by the constant heavy domain CH1 of said antibody, and in the heavy chain the variable heavy domain VH is replaced by the variable light domain VL of said antibody and the constant heavy domain CH1 is replaced by the constant light domain CL of said antibody.

[0021] In one embodiment, a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second antigen-binding domain that specifically binds to PD1 comprises: a) a first light chain and a first heavy chain of an antibody that specifically binds to a first antigen; b) A bivalent antibody comprising a second light chain and a second heavy chain of an antibody which specifically binds to a second antigen, wherein the variable domains VL and VH of the second light chain and the second heavy chain are replaced by each other.

[0022] The two subunits of the antibody of a) do not contain the modifications reported in b), and the heavy and light chains of a) are isolated chains. In the two subunits of the antibody of b), in the light chain the variable light domain VL is replaced by the variable heavy domain VH of said antibody, and in the heavy chain the variable heavy domain VH is replaced by the variable light domain VL of said antibody.

[0023] In one embodiment, a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second antigen-binding domain that specifically binds to PD1 comprises: a) a first light chain and a first heavy chain of an antibody that specifically binds to a first antigen; b) A bivalent antibody comprising a second light chain and a second heavy chain of an antibody which specifically binds to a second antigen, wherein the constant domains CL and CH1 of the second light chain and the second heavy chain are replaced by each other.

[0024] The two subunits of the antibody in a) do not contain the modifications reported in b), and the heavy and light chains of a) are isolated chains. In the two subunits of the antibody in b), the constant light chain domain CL is replaced in the light chain by the constant heavy chain domain CH1 of the antibody, and the constant heavy chain domain CH1 is replaced in the heavy chain by the constant light chain domain CL of the antibody.

[0025] In a further aspect, the invention provides a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second and optionally a third antigen-binding domain that specifically binds to PD1, wherein the first and second antigen-binding domains, and, if present, the third antigen-binding domain, are each Fab fragments, and (i) the second antigen-binding domain is fused at the C-terminus of its Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding domain, or (ii) the first antigen-binding domain is fused at the C-terminus of its Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding domain. In other words, the bispecific antibody is composed of Fab fragments fused to each other. The third antigen-binding domain, if present, is fused to the bispecific antibody at the C-terminus of its Fab heavy chain to the free N-terminus of one of the other two Fab heavy chains, or at the N-terminus of its Fab heavy chain to the free C-terminus of one of the other two Fab heavy chains (see also Figure 4 for exemplary conformations). In one embodiment, the bispecific antibody comprises exactly one (monovalent) antigen-binding domain that specifically binds TfR and exactly two (monovalent) antigen-binding domains that specifically bind PD1.

[0026] In one embodiment, a bispecific antibody comprises a first antigen-binding domain that specifically binds to TfR and a second and a third antigen-binding domain that specifically bind to PD1, said bispecific antibody comprising: a) a full-length antibody consisting of two antibody heavy chains and two antibody light chains and containing two antigen-binding domains that specifically bind to PD1; b) i) an antibody heavy chain variable domain (VH), or ii) an antibody heavy chain variable domain (VH) and an antibody constant domain 1 (CH1) A first polypeptide consisting of a first polypeptide, the first polypeptide being fused at the N-terminus of its VH domain to the C-terminus of one of the two heavy chains of the full-length antibody via a peptidic linker; c) i) an antibody light chain variable domain (VL) or ii) An antibody light chain variable domain (VL) and an antibody light chain constant domain (CL) A second polypeptide consisting of the second polypeptide is a trivalent antibody comprising the N-terminus of the VL domain or the CL domain and a second polypeptide optionally fused to the C-terminus of the other of the two heavy chains of the full-length antibody via a peptidic linker; The antibody heavy chain variable domain (VH) of the first polypeptide and the antibody light chain variable domain (VL) of the second polypeptide together form an antigen-binding domain that specifically binds to the TfR.

[0027] In another embodiment, a bispecific antibody comprises a first antigen-binding domain that specifically binds TfR and a second and a third antigen-binding domain that specifically binds PD1, said bispecific antibody comprising: a) a full-length antibody consisting of two antibody heavy chains and two antibody light chains, the full-length antibody comprising a first antigen-binding domain that specifically binds to PD1, and a second antigen-binding domain that specifically binds to TfR; b) i) an antibody heavy chain variable domain (VH), or ii) an antibody heavy chain variable domain (VH) and an antibody constant domain 1 (CH1) A first polypeptide consisting of a first polypeptide, the first polypeptide being fused to the C-terminus of its VH domain, or, if present, the C-terminus of its constant domain 1 (CH1), via a peptidic linker, to the N-terminus of one of the two heavy chains of the full-length antibody; c) i) an antibody light chain variable domain (VL) or ii) An antibody light chain variable domain (VL) and an antibody light chain constant domain (CL) A second polypeptide consisting of the second polypeptide is a trivalent antibody comprising the C-terminus of the VL domain, or, if present, the C-terminus of the CL domain, and a second polypeptide optionally fused via a peptidic linker to the N-terminus of the other of the two heavy chains of the full-length antibody, The antibody heavy chain variable domain (VH) of the first polypeptide and the antibody light chain variable domain (VL) of the second polypeptide together form an antigen-binding domain that specifically binds to PD1. In certain embodiments, the first and optionally second polypeptides are fused to the N-terminus of a heavy chain that comprises the VH domain of the antigen-binding domain that specifically binds to TfR.

[0028] In one aspect, the invention provides a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second and optionally a third antigen-binding domain that specifically binds to PD1, wherein the first antigen-binding domain, the second antigen-binding domain and, if present, the third antigen-binding domain are each Fab fragments, and the antibody comprises an Fc domain composed of the first and second subunits, (i) a second antigen-binding domain is fused at the C-terminus of its Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding domain, and a first antigen-binding domain is fused at the C-terminus of its Fab heavy chain to the N-terminus of the first subunit of the Fc domain; or (ii) a first antigen-binding domain is fused at the C-terminus of its Fab heavy chain to the N-terminus of the Fab heavy chain of a second antigen-binding domain, and a second antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain; (iii) A bispecific antibody, wherein the third antigen-binding domain, if present, is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain. In one embodiment, the bispecific antibody is a trivalent antibody. In another embodiment, the bispecific antibody comprises exactly one (monovalent) antigen-binding domain that specifically binds to TfR and exactly two (monovalent) antigen-binding domains that specifically bind to PD1. In a particular embodiment, the bispecific antibody is of the IgG class. In another embodiment, the Fab fragment and / or the Fc region of the bispecific antibody is of the IgG class. In a particular embodiment, the bispecific antibody is of the IgG1 isotype. In another embodiment, the Fab fragment and / or the Fc region of the bispecific antibody is of the IgG1 isotype.

[0029] In a further aspect, the invention provides a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second and optionally a third antigen-binding domain that specifically binds to PD1, wherein the first antigen-binding domain, the second antigen-binding domain and, if present, the third antigen-binding domain are each Fab fragments, and the antibody comprises an Fc domain composed of the first and second subunits, i) a first antigen-binding domain is fused at the N-terminus of its Fab heavy chain to the C-terminus of the first or second subunit of the Fc domain, a second antigen-binding domain is fused at the C-terminus of its Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and, if present, a third antigen-binding domain is fused at the C-terminus of its Fab heavy chain to the N-terminus of the second subunit of the Fc domain; or or ii) A bispecific antibody is provided, in which the first antigen-binding domain is fused at the C-terminus of its Fab heavy chain to the N-terminus of the first subunit of the Fc domain, the second antigen-binding domain is fused at the C-terminus of its Fab heavy chain to the N-terminus of the second subunit of the Fc domain, and, if present, the third antigen-binding domain is fused at the N-terminus of its Fab heavy chain to the C-terminus of the first or second subunit of the Fc domain. In one embodiment, the bispecific antibody is a trivalent antibody. In another embodiment, the bispecific antibody comprises exactly one (monovalent) antigen-binding domain that specifically binds to TfR and exactly two (monovalent) antigen-binding domains that specifically bind to PD1. In certain embodiments, the bispecific antibody is of the IgG class. In additional embodiments, the Fab fragment and / or the Fc region of the bispecific antibody is of the IgG class. In certain embodiments, the bispecific antibody is of the IgG1 isotype. In yet another embodiment, the Fab fragment and / or the Fc region of the bispecific antibody is of the IgG1 isotype.

[0030] In one aspect, the bispecific antibody comprises: a) one Fab fragment comprising a first antigen-binding domain that specifically binds to TfR; b) two CrossFab fragments containing an antigen-binding domain that specifically binds to PD1, in which the CH1 and CL domains are exchanged with each other; c) a trivalent antibody comprising one Fc region comprising a first Fc region heavy chain and a second Fc region heavy chain, A trivalent antibody in which the C-terminus of the CH1 domain of the Fab fragment is connected to the N-terminus of one of the heavy chain Fc region polypeptides, the C-terminus of the CH1 domain of one CrossFab fragment is connected to the N-terminus of the other heavy chain Fc region polypeptide, and the C-terminus of the CH1 domain of the other CrossFab fragment is connected to the N-terminus of the VH domain of the Fab fragment or to the N-terminus of the VH domain of the CrossFab fragment. In a particular embodiment, the connection is via a peptidic linker. In another embodiment, the bispecific antibody comprises exactly one (monovalent) antigen-binding domain that specifically binds to TfR and exactly two (monovalent) antigen-binding domains that specifically bind to PD1. In a particular embodiment, the bispecific antibody is of the IgG class. In an additional embodiment, the Fab fragment and / or the Fc region of the bispecific antibody is of the IgG class. In a particular embodiment, the bispecific antibody is of the IgG1 isotype. In yet another embodiment, the Fab fragment and / or the Fc region of the bispecific antibody is of the IgG1 isotype.

[0031] In one embodiment a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second and optionally a third antigen-binding domain that specifically binds to PD1 comprises a) a full-length antibody which specifically binds to PD1 and which consists of two antibody heavy chains and two antibody light chains, in which in the light chain the variable light domain VL is replaced by the variable heavy domain VH of said antibody, and in the heavy chain fragment the variable heavy domain VH is replaced by the variable light domain VL of said antibody, b) a Fab fragment that specifically binds to TfR; The N-terminus of the Fab fragment heavy chain is connected to the C-terminus of one of the two heavy chains of the full-length antibody. In another embodiment, the bispecific antibody comprises exactly one (monovalent) antigen-binding domain that specifically binds to TfR and exactly two (monovalent) antigen-binding domains that specifically bind to PD1. In certain embodiments, the bispecific antibody is of the IgG class. In additional embodiments, the Fab fragment and / or the full-length antibody is of the IgG class. In certain embodiments, the bispecific antibody is of the IgG1 isotype. In yet another embodiment, the Fab fragment and / or the full-length antibody is of the IgG1 isotype.

[0032] In a further embodiment, the bispecific antibody comprises a first antigen-binding domain that specifically binds to TfR, and a second and optionally a third antigen-binding domain that specifically binds to PD1; The first antigen-binding domain that specifically binds to TfR is (a) a heavy chain variable domain (VH) comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO:1, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:2, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:3, and (d) a light chain variable domain (VL) comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:5, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:6; or (a) a heavy chain variable domain (VH) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11; and (d) a light chain variable domain (VL) comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 12, (e) a CDR-L2 having the amino acid sequence of SEQ ID NO: 13, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 14.

[0033] In one embodiment, of a bispecific antibody comprising a first antigen-binding domain that specifically binds TfR and a second and optionally a third antigen-binding domain that specifically binds PD1, the second antigen-binding domain that specifically binds PD1 and / or, if present, the third antigen-binding domain is (a) a heavy chain variable domain (VH) comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 17, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 18, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 19, and (d) a light chain variable domain (VL) comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 20, (e) a CDR-L2 having the amino acid sequence of SEQ ID NO: 21, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 22. or (a) a heavy chain variable domain (VH) comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 25, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 26, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 27, and (d) a light chain variable domain (VL) comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 28, (e) a CDR-L2 having the amino acid sequence of SEQ ID NO: 29, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 30.

[0034] In a particular embodiment, the invention provides a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second and optionally a third antigen-binding domain that specifically binds to PD1, wherein the bispecific antibody simultaneously binds to TfR and PD1, and upon simultaneous binding of the bispecific antibody, a complex formed by the bispecific antibody, TfR and PD1 is internalized into the cell, PD1 is depleted from the cell surface, and the bispecific antibody (a) a heavy chain variable domain (VH) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:1; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:2; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:3; and (d) a light chain variable domain (VL) comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:5, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:6; or (a) a heavy chain variable domain (VH) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11; and (d) a light chain variable domain (VL) comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 12, (e) a CDR-L2 having the amino acid sequence of SEQ ID NO: 13, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 14. A first antigen-binding domain that specifically binds to TfR, comprising: And (a) a heavy chain variable domain (VH) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 17; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 18; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 19; and (d) a light chain variable domain (VL) comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 20, (e) a CDR-L2 having the amino acid sequence of SEQ ID NO: 21, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 22. or (a) a heavy chain variable domain (VH) comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 25, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 26, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 27; and (d) a light chain variable domain (VL) comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 28, (e) a CDR-L2 having the amino acid sequence of SEQ ID NO: 29, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 30. The bispecific antibody further comprises a second antigen-binding domain that specifically binds to PD1 and / or, if present, a third antigen-binding domain comprising:

[0035] In a further embodiment, the bispecific antibody comprises a first antigen-binding domain that specifically binds to TfR, and a second and optionally a third antigen-binding domain that specifically binds to PD1; The first antigen-binding domain that specifically binds to TfR is a VH domain comprising the amino acid sequence of SEQ ID NO: 7 and a VL domain comprising the amino acid sequence of SEQ ID NO: 8; or comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 15 and a VL domain comprising the amino acid sequence of SEQ ID NO: 16; The second antigen-binding domain that specifically binds to PD1 and / or, if present, the third antigen-binding domain a VH domain comprising the amino acid sequence of SEQ ID NO: 23 and a VL domain comprising the amino acid sequence of SEQ ID NO: 24; or It comprises a VH domain comprising the amino acid sequence of SEQ ID NO:31 and a VL domain comprising the amino acid sequence of SEQ ID NO:32.

[0036] In a further embodiment, the bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second and optionally a third antigen-binding domain that specifically binds to PD1 is a monoclonal antibody.

[0037] In certain embodiments, a bispecific antibody comprising a first antigen-binding domain that specifically binds TfR and a second and optionally a third antigen-binding domain that specifically binds PD1 is a humanized antibody or a chimeric antibody.

[0038] In another aspect, the invention provides a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR, and a second and optionally a third antigen-binding domain that specifically binds to PD1, a first heavy chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 35; a first light chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 36; and a second heavy chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 39, and a second light chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 40; or a first heavy chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 37; a first light chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 38; and a second heavy chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 39, and a second light chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 40; or a first heavy chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 35; a first light chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 36; and a second heavy chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 41, and a second light chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 42; or a first heavy chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 37; a first light chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 38; and The present invention relates to a bispecific antibody comprising a second heavy chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 41, and a second light chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 42.

[0039] In another aspect, the invention provides a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second and a third antigen-binding domain that specifically bind to PD1, a first heavy chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO:59, a second heavy chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO:60, a first light chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO:57, and a second light chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO:58; or The present invention relates to a bispecific antibody comprising a first heavy chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 61, a second heavy chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 60, a first light chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 57, and a second light chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 58.

[0040] According to another aspect of the invention, a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second and optionally a third antigen-binding domain that specifically binds to PD1 is an antibody that binds to both TfR and PD1 with an affinity in the nM to sub-nM range as determined by state of the art methods described herein and known to those of skill in the art.

[0041] According to another aspect of the invention, a bispecific antibody comprising a first antigen-binding domain that specifically binds TfR and a second and optionally a third antigen-binding domain that specifically binds PD1 is independently characterized by one or more of the following properties: an anti-PD1 anti-TfR bispecific antibody is i) reduces PD1 / PD-L1 mediated inhibition of TCR signaling by more than 2-fold at a concentration of 1 nM or by more than 4-fold at a concentration of 100 nM (as detected in the co-culture assay according to Example 4 using an NFAT response element operably linked to a luciferase reporter system); and / or ii) upon contact with activated T cells (in an internalization assay according to Example 6), more than 25%, preferably more than 40%, and more preferably more than 50% are internalized into activated T cells iii) (in a minimal mixed lymphocyte reaction according to Example 14) enhances granzyme B secretion by allogeneic stimulated T cells.

[0042] In one embodiment, a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second and optionally a third antigen-binding domain that specifically binds to PD1 is a multispecific antibody.

[0043] In one particular aspect, the invention provides a bispecific antibody comprising a first antigen-binding domain that specifically binds TfR and a second and optionally a third antigen-binding domain that specifically binds PD1, the first antigen-binding domain comprising a first heavy chain of SEQ ID NO: 35, a first light chain of SEQ ID NO: 36, a second heavy chain of SEQ ID NO: 39, and a second light chain of SEQ ID NO: 40.

[0044] In one particular aspect, the invention provides a bispecific antibody comprising a first antigen-binding domain that specifically binds TfR and a second and optionally a third antigen-binding domain that specifically binds PD1, the first antigen-binding domain comprising a first heavy chain of SEQ ID NO: 37, a first light chain of SEQ ID NO: 38, a second heavy chain of SEQ ID NO: 39, and a second light chain of SEQ ID NO: 40.

[0045] In one particular aspect, the invention provides a bispecific antibody comprising a first antigen-binding domain that specifically binds TfR and a second and optionally a third antigen-binding domain that specifically binds PD1, the first antigen-binding domain comprising a first heavy chain of SEQ ID NO: 35, a first light chain of SEQ ID NO: 36, a second heavy chain of SEQ ID NO: 41, and a second light chain of SEQ ID NO: 42.

[0046] In one particular aspect, the invention provides a bispecific antibody comprising a first antigen-binding domain that specifically binds TfR and a second and optionally a third antigen-binding domain that specifically binds PD1, the first antigen-binding domain comprising a first heavy chain of SEQ ID NO: 37, a first light chain of SEQ ID NO: 38, a second heavy chain of SEQ ID NO: 41, and a second light chain of SEQ ID NO: 42.

[0047] In one particular aspect, the invention provides a bispecific antibody comprising a first antigen-binding domain that specifically binds TfR and a second and optionally a third antigen-binding domain that specifically binds PD1, the first antigen-binding domain comprising a first heavy chain of SEQ ID NO: 59, a second heavy chain of SEQ ID NO: 60, a first light chain of SEQ ID NO: 57, and a second light chain of SEQ ID NO: 58.

[0048] In one particular aspect, the invention provides a bispecific antibody comprising a first antigen-binding domain that specifically binds TfR and a second and optionally a third antigen-binding domain that specifically binds PD1, the first antigen-binding domain comprising a first heavy chain of SEQ ID NO: 61, a second heavy chain of SEQ ID NO: 60, a first light chain of SEQ ID NO: 57, and a second light chain of SEQ ID NO: 58.

[0049] In another embodiment, the invention provides an immunoconjugate comprising a bispecific antibody comprising a first antigen-binding domain that specifically binds TfR and a second and optionally a third antigen-binding domain that specifically binds PD1, and a cytotoxic agent. In a particular embodiment, the cytotoxic agent is Pseudomonas exotoxin A or an amatoxin.

[0050] In a further aspect, the bispecific antibody comprises: a) a full-length bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR, and a second and optionally a third antigen-binding domain that specifically binds to PD1, the full-length bispecific antibody consisting of two antibody heavy chains and two antibody light chains; and b) comprises 1, 2, 3 or 4 single chain Fab fragments which specifically bind to 1 to 4 further antigens (i.e. specifically bind to a third and / or a fourth and / or a fifth and / or a sixth antigen, preferably one further antigen, i.e. the third antigen); a) a multispecific antibody, wherein said single-chain Fab fragment of b) is fused to said full-length antibody of a) via a peptidic linker at the C-terminus or N-terminus of the heavy or light chain of said full-length antibody. In a further embodiment, the multispecific antibody comprises exactly one (monovalent) antigen-binding domain that specifically binds to TfR and exactly two (monovalent) antigen-binding domains that specifically bind to PD1.

[0051] In a further aspect, the bispecific antibody comprises: a) a full-length bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR, and a second and optionally a third antigen-binding domain that specifically binds to PD1, the full-length bispecific antibody consisting of two antibody heavy chains and two antibody light chains; and b) comprises one, two, three or four single-chain Fab fragments that specifically bind to biotin; b) A multispecific antibody in which the single-chain Fab fragment is fused to the full-length antibody of a) via a peptidic linker at the C-terminus or N-terminus of the heavy chain or light chain of the full-length antibody.

[0052] In one embodiment, one or two identical single-chain Fab fragments that bind to a third antigen are fused to the full-length antibody at the C-terminus of the heavy or light chain of said full-length antibody via a peptidic linker. In a preferred embodiment, the third antigen is biotin.

[0053] In one embodiment, one or two identical single-chain Fab fragments that bind to a third antigen are fused to the full-length antibody at the C-terminus of the heavy chain of said full-length antibody via a peptidic linker. In a preferred embodiment, the third antigen is biotin.

[0054] In one embodiment, one or two identical single-chain Fab fragments that bind to a third antigen are fused to the full-length antibody at the C-terminus of the light chain of said full-length antibody via a peptidic linker. In a preferred embodiment, the third antigen is biotin.

[0055] In one embodiment, two identical single-chain Fab fragments that bind to a third antigen are fused to the full-length antibody via a peptidic linker at the C-terminus of each heavy or light chain of said full-length antibody. In a preferred embodiment, the third antigen is biotin.

[0056] In one embodiment, two identical single-chain Fab fragments that bind to a third antigen are fused to a full-length antibody at the C-terminus of each heavy chain of said full-length antibody via a peptidic linker.

[0057] In one embodiment, two identical single-chain Fab fragments that bind to a third antigen are fused to a full-length antibody at the C-terminus of each light chain of said full-length antibody via a peptidic linker.

[0058] In one particular embodiment, the present invention provides a trispecific antibody comprising a first antigen-binding domain that specifically binds to TfR, a second antigen-binding domain that specifically binds to PD1, and a third antigen-binding domain that specifically binds to biotin. In one particular embodiment, the present invention provides a trispecific antibody comprising a first antigen-binding domain that specifically binds to TfR, a second antigen-binding domain that specifically binds to PD1, and a third antigen-binding domain that specifically binds to biotin, comprising a first heavy chain of SEQ ID NO: 47, a first light chain of SEQ ID NO: 48, a second heavy chain of SEQ ID NO: 49, and a second light chain of SEQ ID NO: 50. In another embodiment, the third antigen-binding domain of the trispecific antibody, which specifically binds to biotin, is used to attach a payload conjugated to biotin to the trispecific antibody. In a particular embodiment, the payload is a cytotoxic agent, preferably Pseudomonas exotoxin A or amatoxin.

[0059] In one aspect, the invention provides an isolated nucleic acid encoding a bispecific antibody comprising a first antigen-binding domain that specifically binds TfR and a second and optionally a third antigen-binding domain that specifically binds PD1. The invention also provides an isolated nucleic acid encoding an immunoconjugate comprising a first antigen-binding domain that specifically binds TfR and a second and optionally a third antigen-binding domain that specifically binds PD1. In a further aspect, the invention provides a host cell comprising the above nucleic acid.

[0060] In another aspect, the invention relates to a method for producing a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second and optionally a third antigen-binding domain that specifically binds to PD1, or an immunoconjugate comprising said bispecific antibody, comprising culturing a host cell comprising a nucleic acid encoding said bispecific antibody or said immunoconjugate under conditions suitable for expression of the antibody. In a particular aspect, the method further comprises recovering the antibody from the host cell. In a further aspect, the invention also relates to a bispecific antibody produced by such a method.

[0061] In one aspect, the invention provides a pharmaceutical composition comprising a bispecific antibody comprising a first antigen-binding domain that specifically binds TfR and a second and optionally a third antigen-binding domain that specifically binds PD1, or an immunoconjugate comprising said bispecific antibody, and a pharma- ceutically acceptable carrier. In an additional aspect, the invention relates to a pharmaceutical composition comprising a first antigen-binding domain that specifically binds TfR and a second and optionally a third antigen-binding domain that specifically binds PD1, or an immunoconjugate comprising said bispecific antibody, a pharma- ceutically acceptable carrier, and an additional therapeutic agent.

[0062] In another aspect, the present invention provides a bispecific antibody comprising a first antigen-binding domain that specifically binds TfR and a second and optionally a third antigen-binding domain that specifically binds PD1, an immunoconjugate comprising the bispecific antibody, or a pharmaceutical composition comprising the bispecific antibody, for use as a medicament.

[0063] i) modulation of immune responses, e.g., restoration of T cell activity; ii) stimulation of immune responses or functions; iii) prevention or treatment of cancer; iv) delaying the progression of cancer; v) Prolonging survival for patients with cancer; vi) acute infection; vii) chronic and acute viral infections, and / or viii) Other conditions dependent on PD1 expression and PD1-mediated immune regulation Also encompassed by the invention is a bispecific antibody comprising a first antigen-binding domain that specifically binds TfR and a second and optionally a third antigen-binding domain that specifically binds PD1, an immunoconjugate comprising the bispecific antibody, or a pharmaceutical composition comprising the bispecific antibody, for use in

[0064] Immunoconjugates or trispecific antibodies with cytotoxic payloads are also useful for i) Treatment of graft-versus-host disease; and / or ii) Prevention or treatment of autoimmune diseases.

[0065] In another aspect, the invention provides a bispecific antibody comprising a first antigen-binding domain that specifically binds TfR and a second and optionally a third antigen-binding domain that specifically binds PD1, an immunoconjugate comprising the bispecific antibody, or a pharmaceutical composition comprising the bispecific antibody, for use in the prevention or treatment of cancer, wherein the bispecific antibody is administered in combination with a chemotherapeutic agent, radiation and / or other agent for use in cancer immunotherapy.

[0066] Further provided is a method of inhibiting the growth of tumor cells in an individual comprising administering to the individual an effective amount of a bispecific antibody comprising a first antigen-binding domain that specifically binds TfR and a second and optionally a third antigen-binding domain that specifically binds PD1, an immunoconjugate comprising the bispecific antibody, or a pharmaceutical composition comprising the bispecific antibody, to inhibit the growth of tumor cells.

[0067] In a particular aspect, the present invention relates to a bispecific antibody comprising a first antigen-binding domain that specifically binds TfR and a second and optionally a third antigen-binding domain that specifically binds PD1, an immunoconjugate comprising the bispecific antibody, or a pharmaceutical composition comprising the bispecific antibody, in the manufacture of a medicament for the treatment of Cancer, ii. Infection or iii. Graft-versus-host disease.

[0068] Furthermore, the present invention discloses the use of a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second and optionally a third antigen-binding domain that specifically binds to PD1, an immunoconjugate comprising the bispecific antibody, or a pharmaceutical composition comprising the bispecific antibody, in the manufacture of a medicament for: i) Modulation of immune responses, e.g., restoration of T cell activity ii) Stimulation of immune response or function iii) delaying the progression of cancer; and / or iv) Prolonged survival or patients with cancer.

[0069] In one aspect, the invention provides a method of treating an individual having graft-versus-host disease comprising administering to the individual an effective amount of a bispecific antibody comprising a first antigen-binding domain that specifically binds TfR and a second and optionally a third antigen-binding domain that specifically binds PD1, an immunoconjugate comprising the bispecific antibody, or a pharmaceutical composition comprising the bispecific antibody. In an additional aspect, a method of treating an individual having graft-versus-host disease is provided comprising administering to the individual an effective amount of a bispecific antibody comprising a first antigen-binding domain that specifically binds TfR and a second and optionally a third antigen-binding domain that specifically binds PD1, an immunoconjugate comprising the bispecific antibody, or a pharmaceutical composition comprising the bispecific antibody, further comprising administering an additional therapeutic agent to the individual. The additional therapeutic agent is preferably selected from the group consisting of chemotherapeutic agents, checkpoint inhibitors, irradiation, and / or other agents for use in cancer immunotherapy, such as immunocytokines, IL-2 and its variants, IL-7, IL-12, PD1-IL2v, costimulatory molecules such as FAP-4-1BBL / OX40 / CD40, TLR agonists, antibody drug conjugates (ADC), and cytotoxic fusion proteins that can be used as potential "primers" for immunotherapy and for "cold-to-hot" transformation of tumors.

[0070] Further provided is a method of inhibiting PD1 function in an individual, comprising administering to the individual an effective amount of a bispecific antibody comprising a first antigen-binding domain that specifically binds TfR and a second and optionally a third antigen-binding domain that specifically binds PD1, an immunoconjugate comprising the bispecific antibody, or a pharmaceutical composition comprising the bispecific antibody, to inhibit PD1 function. The individual is preferably a mammal, in particular a human. [Brief description of the drawings]

[0071] [Figure 1]Schematic diagram of exemplary configurations of bispecific antibodies of the invention, with a 1+1 stoichiometry of anti-PD1 and anti-TfR specific binding domains. The two different binding domains are distinguished by their patterns. For each configuration, two possible orientations of the charge variants (indicated by ++ or --) that promote heterodimerization are shown, one with the charge in the Fab (top row) and one in the CrossFab (bottom row). (A,F) Diagrams of "1+1 CrossMab VH-VL" molecules. (B,G) Diagrams of "1 arm 1+1 IgG CrossMab VH-VL" molecules with different ordering of CrossFab and Fab components. (C,H) Diagrams of "1 arm 1+1 IgG CrossMab VH-VL" molecules. (D,I) Diagrams of "Fab-CrossFab VH-VL" fusion molecules. (E,J) Diagrams of "CrossFab-Fab VH-VL" fusion molecules. Black dots: any modification in the Fc domain that promotes heterodimerization. ++, --: amino acids of opposite charge that may be introduced into the CH1 and CL domains. Although CrossFab molecules are shown as comprising swapping of VH and VL domains, in embodiments where charge modifications are not introduced into the CH1 and CL domains, they may alternatively comprise swapping of the CH1 and CL domains. [Diagram 2]Schematic diagram of an exemplary configuration of a bispecific antibody of the invention with a 2+1 stoichiometry of anti-PD1 and anti-TfR specific binding domains (one binding domain is attached to the N-terminus of the heavy chain of one Fab ("TCB format")). The different binding domains are distinguished by their pattern. The binding domain that is present twice is the anti-PD1 binding domain. The binding domain that is present once is the TfR binding domain. For each CrossMab configuration, two possible orientations of the charge variants (indicated by ++ or --) that promote heterodimerization are shown, one where the charge is in the Fab (top row) and one where the charge is in the CrossFab (bottom row). (A,E) Diagram of a "2+1 IgG CrossMab VH-VL" molecule. (B,F) Diagram of a "2+1 IgG CrossMab VH-VL" molecule with two CrossFabs and one Fab fused via the C-terminus of its CH1 domain to the N-terminus of the VL domain of one of the CrossFabs. (C, G) Diagram of a "2+1 IgG CrossMab VH-VL" molecule with two CrossFabs and CrossFab and Fab components in different order ("flipped"). (D, H) Diagram of a "2+1 IgG CrossMab" molecule ("flipped"). Black dots: optional modifications in the Fc domain that promote heterodimerization. ++, --: amino acids of opposite charge that may be introduced into the CH1 and CL domains. Although the CrossFab molecule is shown as including an exchange of VH and VL regions, in embodiments where charge modifications are not introduced into the CH1 and CL domains, it may alternatively include an exchange of the CH1 and CL domains. [Diagram 3]Schematic diagram of exemplary configurations of bispecific antibodies of the invention with a 2+1 stoichiometry of anti-PD1 and anti-TfR specific binding domains (one binding domain is attached to the C-terminus of one Fc heavy chain ("BBB format")). The different binding domains are distinguished in this schematic by their pattern. The binding domain present twice is the anti-PD1 binding domain. The binding domain present once is the TfR binding domain. For each configuration, two possible orientations of the charge variants (indicated by ++ or --) that promote heterodimerization are shown, one where the charge is in the Fab (top row) and one where it is in the CrossFab (bottom row). (A, E) Diagrams of "(CrossFab)2-Fc-Fab" molecules. (B, F) Diagrams of "(Fab)2-Fc-CrossFab" molecules. (C, G) Diagrams of "(Fab+CrossFab)-Fc-Fab" molecules. (D, H) Diagram of "(Fab+CrossFab)-Fc-CrossFab" molecule. Black dots: optional modifications in the Fc domain that promote heterodimerization. ++, --: amino acids of opposite charge that may be introduced into the CH1 and CL domains. Although CrossFab molecules are shown as including swapping of VH and VL regions, in embodiments where charge modifications are not introduced into the CH1 and CL domains, they may alternatively include swapping of the CH1 and CL domains. [Figure 4]Schematic diagram of an exemplary configuration of a bispecific antibody of the invention, with a 2+1 stoichiometry of anti-PD1 and anti-TfR specific binding domains (three Fab molecules are covalently linked to each other via peptide linkers as shown). The different binding domains are distinguished by their pattern. The binding domain present twice is the anti-PD1 binding domain. The binding domain present once is the TfR binding domain. For each configuration of the fusion molecule, two possible orientations of the charge variants (indicated by ++ or --) that promote heterodimerization are shown, one where the charge is in the Fab (top row) and one where it is in the CrossFab (bottom row). (A,E) Diagram of a "(Fab)2-Crossfab" molecule. (B,F) Diagram of a "CrossFab-(Fab)2" molecule. (C,G) Diagram of a "(CrossFab)2-Fab". (D,H) Diagram of a "Fab-(CrossFab)2" molecule. ++, --: amino acids of opposite charge that may be introduced into the CH1 and CL domains. Although CrossFab molecules are shown as comprising swapped VH and VL domains, they may alternatively comprise swapped CH1 and CL domains in embodiments where charge modifications are not introduced into the CH1 and CL domains. [Figure 5A] Schematic diagram of the bispecific 1+1 CrossMab with binding domains for TfR and PD1. [Figure 5B] Schematic of a bispecific CrossMab with a third binding domain that specifically binds biotin for payload delivery to activated immune cells. [Figure 6]Schematic diagram of the 2+1 antibody (blood-brain barrier shuttle (BBB) ​​format) used in the examples. The tested 2+1 format antibody molecules 8156 and 8158 were produced as "2+1 IgG CrossMab VH-VL, inverted" with charge modifications (i.e., VH / VL exchange for PD1 binder, charge modifications for TfR binder: EE=147E, 213E; RK=123R, 124K). (B-E) Components for constructing the antibody: light chain of anti-PD1 crossover Fab domain (A), light chain of anti-TfR Fab domain with charge modification in CL (B), heavy chain of anti-PD1 crossover with hole and PG LALA mutation in Fc region and N-terminus of heavy chain of anti-TfR Fab molecule C-terminally linked to Fc region (H), heavy chain with anti-PD1 crossover Fab with knob and PG LALA mutation in Fc region (K). For the control molecule 8158, the light and heavy chain variable antibody regions of the TfR binding arm were replaced by a non-binding sequence ("Nada"). [Figure 7] Schematic diagram of the 2+1 antibodies (T cell bispecific antibody (TCB) format) used in the examples. Tested antibody molecules 8157 and 8159 were produced as "2+1 (CrossFab) 2-Fc-Fab VH-VL" with charge modifications (i.e., VH / VL exchange for PD1 binder, charge modifications for TfR binder: EE=147E, 213E; RK=123R, 124K). (B-E). Components for constructing the antibodies: light chain of anti-PD1 crossover Fab molecule (A), light chain of anti-TfR Fab molecule with charge modification in CL (B), heavy chain of anti-TfR molecule with hole and PG LALA mutation in Fc region and C-terminus of heavy chain of anti-PD1 crossover Fab molecule bound to N-terminus of anti-PD1 Fab (H), heavy chain of anti-PD1 crossover with knob and PG LALA mutation in Fc region (K). For the control molecule 8159, the light and heavy chain variable antibody regions of the TfR binding arm were replaced by a non-binding sequence ("Nada"). [Figure 8A]Blocking PD1 / PD-L1 signaling in co-culture assays. PD1-expressing Jurkat-PD1-NFAT cells were pre-incubated with antibodies for 30 min at 37° C., washed once with medium, and then added to activator cells (PD-L1-expressing CHO-K1 cells that had been allowed to adhere overnight) for 5 h. Inhibition of TCR activation by PD1 signaling was measured by luminescence signal after addition of Bio-Glo™ Luciferase Assay Substrate (representative of three independent experiments). [Figure 8B] Blockade of PD1 / PD-L1 signaling in co-culture assays. Cell viability in the assay after addition of antibodies. Cell viability in the co-culture assays was not affected by the addition of any of the antibodies at the concentrations applied. [Figure 9] SPR curves of the trispecific anti-PD1 anti-TfR anti-biotin CrossMab molecule (1129) and the anti-PD1 anti-Nada anti-biotin control molecule (9904). [Figure 10A] Avidity-enhanced binding of the trispecific anti-PD1 anti-TfR anti-biotin CrossMab is dependent on PD1 expression. Expression levels of TfR and PD1 on PD1-transduced NFAT-bla Jurkat cells analyzed by flow cytometry. [Figure 10B] Avidity-enhanced binding of trispecific anti-PD1 anti-TfR anti-biotin CrossMab is dependent on PD1 expression. Quantification of PE-labeled antibody bound to the surface of PD1-transduced NFAT-bla Jurkat cells (n=3 ± standard error of the mean (SEM)). [Figure 10C]Avidity-enhanced binding of trispecific anti-PD1 anti-TfR anti-biotin CrossMab depends on PD1 expression. CrossMab binding to PD1-transduced NFAT-bla Jurkat cells detected by bio-Cy5 (representative of three independent experiments). Trispecific anti-PD1 anti-TfR anti-biotin CrossMab 1129 and controls were incubated with Jurkat cells expressing different levels of PD1 on their surface (wild type WT, PD1 low, PD1 high). Antibodies were detected using biotinylated Cy5 and by flow cytometry measuring median APC. Binding was stronger for cells that expressed high levels of PD1 on their cell surface. [Figure 11A] Internalization of anti-PD1 anti-TfR CrossMab by activated T cells. Anti-PD1 anti-TfR bispecific antibodies 8012, 8013, 8017 and 8018 show similar internalization as TfR Nada control antibodies (8015, 8016). Antibodies with only the PD1 binding domain but no TfR binding domain show no internalization (PD1-0103-0312, 8014, 8019). [Figure 11B] Internalization of anti-PD1 anti-TfR CrossMab by activated T cells. Anti-PD1 anti-TfR bispecific antibodies 8012, 8013, 8017 and 8018 show similar internalization as TfR Nada control antibodies (8015, 8016). Antibodies with only the PD1 binding domain but no TfR binding domain show no internalization (PD1-0103-0312, 8014, 8019). [Figure 12] Internalization and colocalization of mEGFP-PD1 and Bio-Cy5 payloads in mEGFP-PD1 transduced Jurkat cells. mEGFP-PD1 Jurkat were incubated with 10 nM of anti-TfR / anti-PD1 / anti-biotin trispecific CrossMab conjugated to Bio-Cy5 or control antibody for 3 hours. GFP-PD1 and Bio-Cy5 localization was assessed by confocal microscopy. [Figure 13]Internalization of mEGFP-PD1 into transduced Jurkat cells. mEGFP-PD1 transduced Jurkat cells were incubated with 10 nM pembrolizumab (bivalent anti-PD1 antibody), anti-TfR / anti-PD1 bispecific antibody or anti-CD33 nonbinding control antibody for 60 min. Localization of mEGFP-PD1 was assessed by confocal microscopy. [Figure 14A] Antibody-mediated reduction and restoration of transduced mEGFP-PD1 in Jurkat cells. mEGFP-PD1 transduced Jurkat cells were treated with 10 nM of the trispecific antibody or control molecule and assessed for their median GFP fluorescence after 1, 3, 24 and 48 hours. [Figure 14B] Antibody-mediated reduction and restoration of transduced mEGFP-PD1 in Jurkat cells. Cells were treated with 10 nM of the trispecific antibody for 24 hours to achieve maximum GFP-PD1 downregulation and GFP signal was monitored over 24 hours. [Figure 15A] Avidity-enhanced delivery of biotinylated Pseudomonas exotoxin PE25. Viability of PD1-transduced NFAT-bla Jurkat cells treated for 48 hours with trispecific antibody complexed to bio-PE25 or toxin only control as measured by CellTiter-Glo® assay. [Figure 15B] Avidity-enhanced delivery of biotinylated Pseudomonas exotoxin PE25. Viability of PD1-transduced NFAT-bla Jurkat cells after 48 h treatment with control antibody without bio-PE25. [Figure 16A] Avidity-enhanced binding and internalization in activated human T cells. Binding of trispecific antibodies (anti-CD3 / CD28) to activated T cells detected by biotinylated Cy5. PD1 was quantified to approximately 8000 molecules / cell and TfR to approximately 200 000 molecules / cell. The trispecific antibodies were detected by biotinylated Cy5 at concentrations where the control antibody showed little binding to activated T cells. [Figure 16B]Avidity-enhanced binding and internalization in activated human T cells. Relative fluorescence of anti-IgG on cell surface versus biotinylated-Cy5 data from two donors (t=1 hr vs. t=0 hr) ± standard deviation. After staining for IgG at 0 and 1 hr, internalization was observed with anti-TfR containing antibodies but not with Nada / anti-PD1 antibodies. [Figure 17A] Coexpression of PD1 and TfR on T cells and avidity-enhanced killing of host-infiltrating T cells in a model of graft-versus-host disease (GvHD). Mice engrafted with human PBMCs routinely develop GvHD and eventually die. [Figure 17B] Co-expression of PD1 and TfR on T cells and avidity-enhanced killing of host infiltrating T cells in a model of graft-versus-host disease (GvHD). Cells from mouse spleens were analyzed by flow cytometry and gated on single human CD3 cells. Infiltrating human CD4-positive and human CD8-positive cells were analyzed for expression of TfR and PD1. More than 70% of human T cells (including CD4 and CD8 T cells) detected within mouse spleen cells were double positive for TfR and PD1. [Figure 17C] Co-expression of PD1 and TfR on T cells and avidity-enhanced killing of host-infiltrating T cells in a model of graft-versus-host disease (GvHD). Treatment of spleen cells with anti-PD1 / TfR antibodies conjugated to PE25 demonstrated a 10- to 1000-fold reduction in the number of human T cells in this cell pool. [Figure 18A]Comparison of the various 2+1 formats of bispecific anti-TfR / anti-PD1 antibodies with bivalent binding to PD1 used in the examples, and various control constructs. From left to right, they correspond to (A) molecules 8157, 8156, (B) PD1-0103-0312, 8159 and 8158. "N" indicates the anti-Nada binding domain. The first 2+1 format (anti-TfR binding domain between the anti-PD1 binding domain and the hinge region; "TCB format"; left side) contains one PD1-binding entity as a regular Fab arm in an IgG configuration and a second PD1-binding Fab arm at the top (i.e., N-terminus) of the TfR-binding CrossFab preceding the hinge on the other side of the knob-into-hole heterodimer. The second 2+1 format (anti-TfR binding domain fused c-terminally to the CH3 of the Fc domain; "BBB format"; right) contains a regular Fab arm of IgG as the PD1-binding arm, and the TfR-binding Fab is attached to the C-terminus of an asymmetric (knob-into-hole) CH3 domain in a CrossFab format. [Figure 18B] Comparison of the various 2+1 formats of bispecific anti-TfR / anti-PD1 antibodies with bivalent binding to PD1 used in the examples, and various control constructs. From left to right, they correspond to (A) molecules 8157, 8156, (B) PD1-0103-0312, 8159 and 8158. "N" indicates the anti-Nada binding domain. On the left side, a "classical" bivalent blocking anti-PD1 antibody (anti-PD1-IgG) is shown. To compare the effect of TfR binding and internalization on anti-PD1 antibodies, two controls were constructed in which TfR was replaced with a non-antigen binding Fab fragment (anti-Nada binding domain between the anti-PD1 binding domain and the hinge region, and anti-Nada binding domain c-terminally fused to CH3 of the Fc domain). [Figure 19]Internalization of two bispecific antibodies, 8156 and 8157 (different 2+1 formats), that bind PD1 in a bivalent manner and TfR in a monovalent manner, compared to anti-PD1 antibodies and two PD1 Nada control antibodies. Control molecules that bind PD1 in a bivalent manner but do not contain a TfR binder (anti-PD1, 8158, 8159) showed significantly lower internalization, whereas the two 2+1 bispecific antibody formats that bind PD1 in a bivalent manner and TfR in a monovalent manner (8157, 8156) showed significantly increased internalization rates. [Figure 20A] Effect of the tested antibodies on cytotoxic granzyme B release by human CD4 T cells co-cultured with allogeneic mature dendritic cells (mixed lymphocyte reaction). The EC50 values ​​achieved by the anti-PD1 anti-Tfr bispecific antibodies 8012 and 8013 are comparable to those achieved by the bivalent PD1-0103-0312 binder. The monovalent anti-PD1 constructs PD1-0103-0312 / Nada (8014) and pembrolizumab / Nada (8019) only resulted in moderate granzyme B secretion, and monovalent TfR binding by binders Nada / 51A165 (8015) and Nada / 1026 (8016) did not induce any granzyme B secretion. [Figure 20B] Effect of the tested antibodies on cytotoxic granzyme B release by human CD4 T cells co-cultured with allogeneic mature dendritic cells (mixed lymphocyte reaction). The EC50 values ​​achieved by the anti-PD1 anti-Tfr bispecific antibodies 8012 and 8013 are comparable to those achieved by the bivalent PD1-0103-0312 binder. The monovalent anti-PD1 constructs PD1-0103-0312 / Nada (8014) and pembrolizumab / Nada (8019) only resulted in moderate granzyme B secretion, and monovalent TfR binding by binders Nada / 51A165 (8015) and Nada / 1026 (8016) did not induce any granzyme B secretion. [Figure 21]Effect of tested antibodies on cytotoxic granzyme B release by human CD4 T cells co-cultured with allogeneic mature dendritic cells (mixed lymphocyte reaction). The TCB and BBB formats (8156 and 8157) showed lower EC50 values ​​than the bivalent parental anti-PD1 antibodies and also resulted in increased granzyme B secretion and therefore increased T cell effector function induced by these formats compared to their respective controls (8158 and 8159). [Figure 22A] Effect of tested antibodies on cytotoxic granzyme B release by human CD4 T cells co-cultured with allogeneic mature dendritic cells (mixed lymphocyte reaction). The EC50 values ​​achieved by the monovalent anti-PD1 anti-Tfr bispecific antibodies 8012 and 8013 were comparable to those achieved by the bivalent PD1-0103-0312 binder. On the other hand, the monovalent anti-PD1 construct PD1-0103-0312 / Nada (8014) only resulted in a moderate granzyme B secretion. [Figure 22B] Effect of tested antibodies on cytotoxic granzyme B release by human CD4 T cells co-cultured with allogeneic mature dendritic cells (mixed lymphocyte reaction). The EC50 values ​​achieved by the monovalent anti-PD1 anti-Tfr bispecific antibodies 8017 and 8018 were comparable to that achieved by pembrolizumab. On the other hand, the monovalent anti-PD1 construct pembrolizumab / Nada (8019) only resulted in a moderate granzyme B secretion. [Figure 22C] Effect of tested antibodies on cytotoxic granzyme B release by human CD4 T cells co-cultured with allogeneic mature dendritic cells (mixed lymphocyte reaction). The EC50 values ​​achieved by the bivalent anti-PD1 anti-Tfr bispecific antibodies 8157 (TCB format) and 8156 (BBB format) were lower than those achieved with pembrolizumab or the bivalent PD1-0103-0312 binder. On the other hand, the bivalent anti-PD1 constructs PD1-0103-0312 / Nada TCB format (8159) and BBB format (8158) only resulted in moderate granzyme B secretion. [Diagram 23] The BA / F3 cell line (RNCB accession ID: CL003201), which expresses mouse TfR on its cell surface, was used to test the internalization of anti-PD1 anti-TfR mouse molecules. Both molecules containing the TfR binding domains 6768 (mTfR-001 / huPD1-478 TCB format) and 6794 (mTfR-001 / Nada TCB format) showed good internalization rates of about 70% after 3 hours, whereas huPD1 / Nada (P1AG6769) showed no internalization. [Figure 24] Anti-PD1 anti-TfR murine molecules were tested for blocking PD1 / PD-L1 mediated signaling in a co-culture assay. Anti-PD1 antibody PD1-0103-0312-like molecules 6768 (mTfR-001 / huPD1-478, TCB format) and 6769 (Nada / huPD1-478, TCB format) contain bivalent anti-PD1 binding domains. All of these molecules showed comparable functionality in blocking the PD1-PDL1 signaling pathway. The control molecule 6794 (mTfR-001 / Nada, TCB format), which does not contain the anti-PD1 antigen binding domain, did not show any blocking functionality. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0072] In one aspect, the invention is based in part on the discovery that selected anti-TfR anti-PD1 bispecific antibodies of the invention have the ability to bind to TfR and PD1 and be internalized into cells that express and display TfR and PD1 on their surface.

[0073] In a further aspect, the invention is based in part on the discovery that anti-TfR anti-PD1 2+1 format antibodies, i.e., bispecific antibodies comprising a first antigen-binding domain that specifically binds TfR and a second and third antigen-binding domain that specifically binds PD1, exhibit improved biological activity over monospecific bivalent PD1 antibodies and provide better inhibition of the interaction between PD1 and PD-L1. In one aspect, these molecules comprise an IgG class Fab fragment and optionally an IgG class Fc region covalently linked to each other, resulting in the different conformations of the 2+1 format antibodies described herein.

[0074] It has been found that contacting cells expressing PD1 and TfR with the anti-TfR anti-PD1 bispecific antibody of the present invention depletes PD1 from the surface of these cells, particularly T cells, thus preventing the binding of PD-L1 to the PD1 receptor on the surface of these cells. The anti-TfR anti-PD1 bispecific antibody inhibits PD1 / PD-L1-mediated T cell receptor signaling, for example, increasing immune-modulating cytokines (e.g., interferon gamma and granzyme B release / secretion). The inhibition achieved by depletion of PD-1 from the cell surface is more effective and / or permanent than the inhibition that can be achieved by the transient binding of anti-PD1 blocking antibodies. Other immune-modulating cytokines that can be increased by using the antibodies of the present invention are, for example, tumor necrosis factor alpha (TNF alpha) secretion and IL-12. As used herein, terms such as interferon gamma (IFN-gamma), tumor necrosis factor alpha (TNF alpha), IL-12, etc. refer to human cytokines.

[0075] I. Definition An "acceptor human framework" for the purposes of this specification is a framework that comprises the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence or may contain amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0076] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding domain of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y is generally determined by the dissociation constant (K D ) Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative exemplary methods for measuring binding affinity are described below.

[0077] An "affinity matured" antibody refers to an antibody that has one or more modifications in one or more complementarity determining regions (CDRs), compared to a parent antibody that does not possess the modifications, which improve the affinity of the antibody for antigen.

[0078] The terms "anti-TfR antibody" and "antibody or antigen binding domain that specifically binds to TfR" refer to an antibody or antigen binding domain that can bind to TfR with sufficient affinity such that the antibody or antigen binding domain is useful as a diagnostic and / or therapeutic agent in targeting TfR. In one embodiment, the degree of binding of an anti-TfR antibody or antibody or antigen binding domain that specifically binds to TfR to an unrelated non-TfR protein is less than about 10% of the binding of the anti-TfR antibody or antigen binding domain to TfR, as measured, for example, by surface plasmon resonance (SPR). In certain embodiments, an antibody comprising an antigen binding domain that binds to TfR has an affinity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g., 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13 The antibody or antigen-binding domain has a dissociation constant (KD) of 1 μM or less. D An anti-TfR antibody is said to "specifically bind" to a TfR if it has an epitope that is conserved among TfRs from different species.

[0079] The terms "anti-PD1 antibody" and "antibody or antigen-binding domain that specifically binds to PD1" refer to an antibody or antigen-binding domain that can bind to PD1 with sufficient affinity such that the antibody or antigen-binding domain is useful as a diagnostic and / or therapeutic agent in targeting PD1. In one embodiment, the extent of binding of an antibody or antigen-binding domain that specifically binds to PD1 to an unrelated non-PD1 protein is less than about 10% of the binding of the antibody or antigen-binding domain to PD1, as measured, for example, by surface plasmon resonance (SPR). In certain embodiments, an antibody or antigen-binding domain that binds to PD1 has an affinity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g., 10 -8 M~10-13 M, for example, 10 -9 M~10 -13 Dissociation constant (K D The antibody or antigen-binding domain has a K D An anti-PD1 antibody or antibody or antigen-binding domain that specifically binds to PD1 is said to "specifically bind" to PD1. In certain embodiments, an anti-PD1 antibody or antibody or antigen-binding domain that specifically binds to PD1 binds to an epitope of PD1 that is conserved among PD1 from different species. The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies, (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity. Various patent applications disclose methods of enhancing immune responses using the production of anti-PD1 antibodies and / or agents that interfere with PD-L1 binding and / or PD1 signaling, including anti-PD1 antibodies, including: U.S. Patent No. 2003 / 0039653, U.S. Patent No. 2004 / 0213795, U.S. Patent No. 2006 / 0110383, U.S. Patent No. 2007 / 0065427, U.S. Patent No. 2007 / 0122378, U.S. Patent No. 2012 / 237522, WO 2004 / 072286, WO 2006 / 121168, WO 2006 / 133396, WO 2007 / 005874, WO 2007 / 005875, WO 2007 / 005711, WO 2007 / 005912, WO 2007 / 005913, WO 2007 / 005914, WO 2007 / 005915, WO 2007 / 005916, WO 2007 / 005917, WO 2007 / 005919 ... International Publication No. 2008 / 083174, International Publication No. 2008 / 156712, International Publication No. 2009 / 024531, International Publication No. 2009 / 014708, International Publication No. 2009 / 114335, International Publication No. 2010 / 027828, International Publication No. 2010 / 027423, International Publication No. 2010 / 036959, International Publication No. WO 2010 / 029435, WO 2010 / 029434, WO 2010 / 063011, WO 2010 / 089411, WO 2011 / 066342, WO 2011 / 110604, WO 2011 / 110621 and WO 2012 / 145493.

[0080] "Blocking" or "antagonist" antibodies are antibodies that inhibit or reduce the biological activity of the antigen to which they bind. In some embodiments, blocking or antagonist antibodies substantially or completely inhibit the biological activity of the antigen. For example, the bispecific antibodies of the invention block signaling by PD1 and PD-L1 to restore functional responses (e.g., proliferation, cytokine production, target cell death) by T cells from dysfunctional states in response to antigenic stimulation.

[0081] The term "monospecific" antibody as used herein means an antibody having one or more binding domains that each bind to the same epitope of the same antigen. The term "bispecific" means that an antibody can specifically bind to at least two distinct antigenic determinants, for example, via two binding domains formed by a pair of antibody heavy chain variable domains (VH) and antibody light chain variable domains (VL) that bind to different antigens or different epitopes on the same antigen. Such bispecific antibodies are also referred to herein as 1+1 format antibodies. Other bispecific antibody formats are referred to herein as 2+1 format antibodies (comprising two binding domains for a first antigen or epitope and one binding domain for a second antigen or epitope) or 2+2 format antibodies (comprising two binding domains for a first antigen or epitope and two binding domains for a second antigen or epitope).

[0082] The terms "a bispecific antibody comprising a first antigen-binding domain that specifically binds TfR and a second and optionally a third antigen-binding domain that specifically binds PD1", "a bispecific antibody that specifically binds TfR and PD1", "a bispecific antigen-binding molecule specific for TfR and PD1" and "an anti-TfR anti-PD1 bispecific antibody" are used interchangeably herein and refer to a bispecific antibody that is capable of binding to TfR and PD1 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting TfR and PD1.

[0083] The term "valency" as used in this application refers to the presence of a certain number of binding domains in an antigen-binding molecule. Thus, the terms "bivalent", "tetravalent" and "hexavalent" refer to the presence of two, four and six binding domains, respectively, in an antigen-binding molecule. A bispecific antibody according to the invention is at least "bivalent" and may be "trivalent" or "multivalent" (e.g., "tetravalent" or "hexavalent"). In certain embodiments, an antibody of the invention has two or more binding domains and is bispecific. That is, an antibody may be bispecific even if more than two binding domains are present (i.e., the antibody is trivalent or multivalent). In particular, the present invention relates to bispecific bivalent and trivalent antibodies having one or two binding domains for each antigen that it specifically binds to.

[0084] The terms "full-length antibody", "intact antibody" and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a natural antibody structure. "Natural antibody" refers to naturally occurring immunoglobulin molecules with various structures. For example, a natural IgG class antibody is a heterotetrameric glycoprotein of about 150,000 Da (Daltons) and is composed of two light chains and two heavy chains that are disulfide-bonded. The individual polypeptide chains that form the antibodies, antibody fragments and antibody-like molecules disclosed herein are sometimes referred to herein as "subunits", e.g., subunits of Fab fragments or subunits of Fc domains. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2 and CH3), also called heavy chain constant region. Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called the variable light domain or light chain variable domain, followed by a light chain constant domain (CL), also called the light chain constant region. The heavy chain of an antibody may be assigned to one of five types, called α (IgA), δ (IgD), ε (IgE), γ (IgG) or μ (IgM), some of which may be further classified into subtypes, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1) and α2 (IgA2). The light chain of an antibody may be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

[0085] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv and scFab); single domain antibodies (dAbs); and multispecific antibodies formed from antibody fragments. For a review of specific antibody fragments, see Holliger and Hudson, Nature Biotechnology 23:1126-1136 (2005). For a review of scFv fragments, see, e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994). See also WO 93 / 16185 and U.S. Patent Nos. 5,571,894 and 5,587,458. For a description of Fab and F(ab')2 fragments that contain salvage receptor binding epitope residues and have increased half-life in vivo, see U.S. Patent No. 5,869,046. Diabodies are antibody fragments that have two antigen-binding domains that can be bivalent or bispecific, see, for example, EP 404,097; WO 1993 / 01161; Hudson et al., Nat Med 9, 129-134 (2003); and Hollinger et al., Proc Natl Acad Sci USA 90, 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat Med 9, 129-134 (2003). Single domain antibodies are antibody fragments that contain all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, single domain antibodies are human single domain antibodies (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516).In addition, antibody fragments contain single-chain polypeptides characteristic of a VH domain (i.e., capable of being assembled with a VL domain) or characteristic of a VL domain (i.e., capable of being assembled with a VH domain into a functional antigen-binding domain), thereby providing antigen-binding properties of a full-length antibody. Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., E. coli or phages), as described herein.

[0086] Papain digestion of an intact antibody produces two identical antigen-binding fragments, called "Fab" fragments, each of which contains the heavy and light chain variable domains, as well as the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Thus, as used herein, the term "Fab fragment" refers to an antibody fragment that contains a light chain fragment containing the VL domain and constant domain (CL) of the light chain, and the VH domain and first constant domain (CH1) of the heavy chain, and contains three CDRs in the VH and three CDRs in the VL. The terms "Fab", "Fab fragment", "Fab molecule" and "Fab domain" are used interchangeably herein to refer to an antibody fragment that contains the heavy and light chain variable domains, the constant domain of the light chain and the first constant domain (CH1) of the heavy chain.

[0087] Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is a Fab' fragment in which the cysteine ​​residues of the constant domains bear a free thiol group. Pepsin treatment yields an F(ab')2 fragment that has two antigen-binding sites (two Fab fragments) and part of the Fc region.

[0088] The term "CrossFab" or "Cross-Fab fragment" or "xFab fragment" or "crossover Fab fragment" refers to a Fab fragment in which either the variable or constant regions of the heavy and light chains have been exchanged. The terms "fragment", "molecule" and "domain" are also used interchangeably herein for these Fab fragment variants and for the Fab fragments described in the following paragraphs. Two different chain compositions of crossover Fab fragments are possible and are included in the bispecific antibodies of the invention: on the one hand, the variable regions of the Fab heavy and Fab light chains are exchanged, i.e. the crossover Fab fragment comprises a peptide chain composed of the light chain variable region (VL) and the heavy chain constant region (CH1) and a peptide chain composed of the heavy chain variable region (VH) and the light chain constant region (CL). This crossover Fab fragment is also called CrossFab (VLVH). On the other hand, when the constant regions of the Fab heavy chain and the Fab light chain are exchanged, the crossover Fab fragment comprises a peptide chain composed of the heavy chain variable region (VH) and the light chain constant region (CL) and a peptide chain composed of the light chain variable region (VL) and the heavy chain constant region (CH1). This crossover Fab fragment is also called CrossFab (CLCH1). As described further herein, to further reduce the mispairing of the heavy and light chains of different Fab molecules and thus increase the purity and yield of the desired (bispecific) antibody, charged amino acids with opposite charges may be introduced into specific amino acid positions in the CH1 and CL domains of the Fab molecule that binds the first antigen (TfR) or the Fab molecule that binds the second antigen (PD1). Charge modifications may be made to either conventional Fab molecules (e.g., as shown in Figures 1A-E, 2A-D, 3A-D, 4A-D) contained in the (bispecific) antibody, or to VH / VL crossover Fab molecules (e.g., as shown in Figures 1F-J, 2E-H, 3E-H, 4E-H) contained in the (bispecific) antibody, but not to both.

[0089] A "single-chain Fab fragment" or "scFab" is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, the antibody domains and the linker having one of the following orders from N-terminus to C-terminus: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, (c) VL-CL-linker-VH-CH1, or d) VL-CH1-linker-VH-CL, the linker being a polypeptide of at least 30 amino acids, preferably 32-50 amino acids. The single-chain Fab fragment is stabilized by a native disulfide bond between the CL and CH1 domains. In addition, these single-chain Fab molecules may be further stabilized by the insertion of cysteine ​​residues (e.g., at position 44 of the variable heavy chain and position 100 of the variable light chain, according to Kabat numbering) to generate interchain disulfide bonds.

[0090] A "crossover single chain Fab fragment" or "x-scFab" is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, said antibody domains and said linker having one of the following orders from N-terminus to C-terminus: a) VH-CL-linker-VL-CH1, and b) VL-CH1-linker-VH-CL, VH and VL together form an antigen binding domain that specifically binds to an antigen, and said linker is a polypeptide of at least 30 amino acids. In addition, these x-scFab molecules may be further stabilized by the creation of an interchain disulfide bond by the insertion of cysteine ​​residues (e.g., at position 44 of the variable heavy chain and position 100 of the variable light chain according to the Kabat numbering).

[0091] A "single chain variable fragment (scFv)" is a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of an antibody, connected by a short linker peptide of 10 to about 25 amino acids. The linker is usually rich in glycine for flexibility and rich in serine or threonine for solubility, and may connect the N-terminus of the VH to the C-terminus of the VL, or vice versa. The protein retains the specificity of the original antibody despite the removal of the constant regions and the introduction of the linker. scFv antibodies are described, for example, in Houston, JS, Methods in Enzymol. 203 (1991) 46-96). In addition, the antibody fragment contains a single chain polypeptide characteristic of the VH domain (i.e., capable of being assembled together with the VL domain) or characteristic of the VL domain (i.e., capable of being assembled together with the VH domain into a functional antigen-binding domain), thereby providing the antigen-binding properties of a full-length antibody.

[0092] "Scaffold antigen-binding proteins" are known in the art, for example, fibronectin and designed ankyrin repeat proteins (DARPins) have been used as alternative scaffolds for antigen-binding domains, see, e.g., Gebauer and Skerra, Engineered protein scaffolds as next-generation antibody therapeutics. Curr Opin Chem Biol 13:245-255 (2009) and Stumpp et al., Darpins: A new generation of protein therapeutics. Drug Discovery Today 13:695-701 (2008). In one embodiment of the invention, the scaffold antigen binding protein is selected from the group consisting of CTLA-4 (Evibodies), lipocalins (Anticalins), Protein A derived molecules such as the Z-domain of Protein A (Affibodies), A-domain (Avimers / Maxibodies), serum transferrin (Transbodies); designed ankyrin repeat proteins (DARPins), variable domains of antibody light or heavy chains (single domain antibodies, sdAbs), variable domains of antibody heavy chains (nanobodies, aVH), VNAR fragments, fibronectin (Adnectins), C-type lectin domains (Tetranectins); variable domains of the novel antigen receptor beta-lactamase (VNAR fragments), human gamma-crystallin or ubiquitin (Affilin molecules); Kunitz-type domains of human protease inhibitors, microbodies such as proteins from the knottin family, peptide aptamers and fibronectin (Adnectins).

[0093] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remaining portions of the heavy and / or light chain are derived from a different source or species.

[0094] The "class" of an antibody refers to the type of constant domain or constant region carried by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In certain embodiments, the antibody is of the IgG class. IgG class antibodies and IgG-like antibody molecules are generally easy to produce and purify in large quantities, and often have pharmacological properties similar to those of conventional IgG1. In certain embodiments, the antibody is of the IgG1 isotype. In certain embodiments, the antibody is of the IgG1 isotype with P329G, L234A, and L235A mutations to reduce Fc region effector function. In other embodiments, the antibody is of the IgG2 isotype. In certain embodiments, the antibody is of the IgG4 isotype with S228P mutation in the hinge region to improve the stability of the IgG4 antibody. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The light chain of an antibody may be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

[0095] The term "constant region of human origin" or "human constant region" as used in this application refers to the constant heavy chain region of a human antibody of subclass IgG1, IgG2, IgG3 or IgG4, and / or the constant light chain kappa or lambda region. Such constant regions are known in the art and are described, for example, in Kabat, EA, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) (see also, for example, Johnson, G., and Wu, TT, Nucleic Acids Res. 28 (2000) 214-218; Kabat, EA, et al., Proc. Natl. Acad. Sci. USA 72 (1975) 2785-2788). Unless otherwise specified herein, numbering of amino acid residues in the constant region follows the EU numbering system (also called Kabat's EU index), as described in Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.

[0096] The term "depletion" as used herein refers to a significant reduction in the number of receptor molecules displayed on the surface of a cell expressing the receptor molecule when the cell is contacted with the antibody of the present invention. Depletion is expressed as the ratio of receptor molecules on the cell surface compared to the number of receptor molecules present on the surface of a control cell that has not been contacted with the antibody of the present invention. Cells in which receptor molecules have been depleted from the cell surface preferably have a greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% reduction, more preferably a greater than 95%, 98% or 99% reduction in the number of receptor molecules displayed on the cell surface compared to untreated control cells. These ranges can be determined by state of the art methods described herein and known to those skilled in the art.

[0097] "Effector function" refers to the biological activity attributable to the Fc region of an antibody, which varies depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

[0098] An "effective amount" of an agent, eg, a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0099] The term "Fc region" is used herein to define the C-terminal region of an IgG class immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the antibody produced by the host cell may undergo post-translational truncation of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Thus, the antibody produced by the host cell by expression of a particular nucleic acid molecule that encodes a full-length heavy chain may include a full-length heavy chain or a truncated variant of the full-length heavy chain. This may be the case when the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, EU numbering system). Thus, the C-terminal lysine (Lys447) or the C-terminal glycine (Gly446) and lysine (Lys447) of the Fc region may be present or absent. The amino acid sequence of the heavy chain comprising the Fc region is shown herein without the C-terminal glycine-lysine dipeptide, unless otherwise indicated. In one embodiment, the heavy chain comprising the Fc region as specified herein, which is comprised in the antibody according to the invention, comprises an additional C-terminal glycine-lysine dipeptide (G446 and K447, EU numbering system). In one embodiment, the heavy chain comprising the Fc region as specified herein, which is comprised in the antibody according to the invention, comprises an additional C-terminal glycine residue (G446, EU index numbering). Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system (also called the EU index), as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0100] "Framework" or "FR" refers to variable domain residues other than the complementarity determining regions (CDRs). The FRs of a variable domain typically consist of four FR domains: FR1, FR2, FR3 and FR4. Thus, the CDR and FR sequences typically appear in the following order in a VH (or VL): FR1-CDR-H1 (CDR-L1)-FR2-CDR-H2 (CDR-L2)-FR3-CDR-H3 (CDR-L3)-FR4.

[0101] According to the Kabat numbering system, as used herein, the framework and CDR regions are located in the following regions of the variable domain: TIFF2024528217000001.tif26170*CDR-H1 may have additional amino acids between positions 35b and 36, referred to herein as positions "35c", "35d" and "35e".

[0102] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a heavy chain having a structure substantially similar to a native antibody structure or containing an Fc region as defined herein.

[0103] The terms "host cell", "host cell line" and "host cell culture" are used interchangeably to refer to such cells, including the progeny of cells into which exogenous nucleic acid has been introduced. Host cells include "transformants" and "transformed cells", which include the primary transformed cell and its progeny, regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the original transformed cell are included herein.

[0104] A "human antibody" is an antibody having an amino acid sequence that corresponds to that of an antibody produced by a human or human cell, or derived from a non-human source that utilizes a human antibody repertoire or other human antibody coding sequence. This definition of a human antibody specifically excludes humanized antibodies, which contain non-human antigen-binding residues.

[0105] A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is derived from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup kappa I as in Kabat et al., supra. In one embodiment, for VH, the subgroup is subgroup III as in Kabat et al., supra.

[0106] A "humanized" antibody refers to a chimeric antibody that comprises amino acid residues from non-human CDRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, with all or substantially all of the CDRs corresponding to the CDRs of a non-human antibody and all or substantially all of the FRs corresponding to the FRs of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has been subjected to humanization.

[0107] The term "hypervariable region" or "HVR" as used herein refers to each region of an antibody variable domain which is hypervariable in sequence and which determines antigen-binding specificity, e.g., the "complementarity determining regions" ("CDRs").

[0108] Generally, an antibody comprises six CDRs: three in the VH (CDR-H1, CDR-H2, CDR-H3) and three in the VL (CDR-L1, CDR-L2, CDR-L3). Exemplary CDRs herein include the following: (a) hypervariable loops located at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs located at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and (c) Antigenic contacts present at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262:732-745 (1996)). TIFF2024528217000002.tif67170

[0109] Unless otherwise indicated, CDRs are determined according to Kabat et al., supra. Those skilled in the art will understand that the designations of CDRs can also be determined according to Chothia, supra, McCallum, supra, or any other scientifically accepted nomenclature system.

[0110] The terms "cell surface receptor", "membrane receptor" and "transmembrane receptor" are used interchangeably herein. Cell surface receptors are specialized integral membrane proteins that allow communication between cells and the extracellular space. They are embedded in the plasma membrane of cells and act in cell signaling and signal transduction by binding to extracellular molecules such as cytokines, growth factors, cell adhesion molecules, hormones, neurotransmitters, nutrients, and by triggering responses within the cell through a series of molecular switches to internal signaling pathways. PD1 and TfR are examples of such cell surface receptors.

[0111] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, including, but not limited to, a cytotoxic agent.

[0112] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs and horses), primates (e.g., humans and non-human primates, e.g., monkeys), rabbits and rodents (e.g., mice and rats). In certain embodiments, an individual or subject is a human.

[0113] The term "internalization" or "receptor internalization" as used herein refers to a biological process also called endocytosis, i.e., a process in which cells ingest molecules (such as proteins) by phagocytosis, resulting in the transport of the molecules from the outside to the inside of the cell. Internalized molecules can be located in intracellular compartments, such as vacuoles, endosomes, lysosomes, endoplasmic reticulum, Golgi apparatus, or cytosol. An "internalized" or "internalizing" antibody refers to an antibody that can be transported from the outside to the inside of a target cell, for example, by binding to an internalizing cell surface receptor, such as the transferrin receptor.

[0114] An "isolated" antibody is an antibody that has been separated from a component of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, for example, as determined by electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse-phase HPLC) methods. For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0115] The terms "linker peptide", "peptide linker" or "peptidic linker" are used interchangeably and refer to short to medium length polypeptides, preferably between 10 and about 25 amino acids. Linker peptides are usually rich in glycine for flexibility and rich in serine or threonine for solubility. "Linker peptide", "peptide linker" or "peptidic linker" refers to a synthetic amino acid sequence that connects or joins two polypeptide sequences, e.g., joins two polypeptide domains. As used herein, the term "synthetic" refers to an amino acid sequence that does not occur in nature. The linker peptides of the present invention connect two amino acid sequences via a peptide bond. Typically, the linker peptide connects a biologically active moiety to a second portion of a linear sequence. In the context of a polypeptide, a "linear sequence" or "sequence" is the order of amino acids in a polypeptide from the amino terminus to the carboxyl terminus in which residues adjacent to each other in the sequence are in close proximity in the primary structure of the polypeptide. As used herein, the terms "linked", "connected", "covalently bonded", "fused" or "fusion" are used interchangeably. In one embodiment, the linker consists primarily or entirely of Gly and Ser. In a further embodiment, the linker has the sequence of SEQ ID NO:108 or SEQ ID NO:109.

[0116] The term "nucleic acid molecule" or "polynucleotide" includes any compound and / or substance that comprises a polymer of nucleotides. Each nucleotide is composed of a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Nucleic acid molecules are often described by the sequence of bases, whereby the bases represent the primary (linear) structure of the nucleic acid molecule. The sequence of bases is typically represented from 5' to 3'. As used herein, the term nucleic acid molecule encompasses, for example, deoxyribonucleic acid (DNA), including complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers that include two or more of these molecules. Nucleic acid molecules can be linear or circular. In addition, the term nucleic acid molecule includes both sense and antisense strands, and single-stranded and double-stranded forms. Furthermore, the nucleic acid molecules described herein can contain natural or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derivatized sugars or phosphate backbone linkages, or chemically modified residues. Nucleic acid molecules also include DNA and RNA molecules suitable as vectors for direct expression of the antibody of the present invention in vitro and / or in vivo, for example, in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors can be unmodified or modified. For example, the mRNA can be chemically modified to enhance the stability of the RNA vector and / or expression of the encoded molecule, such that the mRNA can be injected into a subject to generate antibodies in vivo (see, e.g., Stadler et al, Nature Medicine 2017, published online 12 June 2017, doi:10.1038 / nm.4356 or European Patent No. 2 101 823).

[0117] An "isolated" nucleic acid is a nucleic acid molecule that is separated from a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule contained in a cell that ordinarily contains the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0118] "Isolated nucleic acid encoding an anti-TfR antibody or an anti-PD1 antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of an anti-TfR antibody or an anti-PD1 antibody, including such nucleic acid molecules in a single vector or separate vectors, and such nucleic acid molecules are present in one or more locations within a host cell.

[0119] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies constituting the population are identical and / or bind to the same epitope, except for variant antibodies that may contain naturally occurring mutations or arise during the production of a monoclonal antibody preparation (e.g., such variants are generally present in minor amounts). In contrast to polyclonal antibody preparations, which typically contain different antibodies against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed to a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody obtained from a population of substantially homogeneous antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies according to the present invention can be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin locus, such methods, as well as other exemplary methods for producing monoclonal antibodies, are described herein.

[0120] "Naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The naked antibody may be present in a pharmaceutical composition.

[0121] "Natural antibody" refers to naturally occurring immunoglobulin molecules with various structures. For example, a natural IgG antibody is a heterotetrameric glycoprotein of about 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable domain (VH), also called variable heavy chain domain or heavy chain variable region, followed by three constant heavy chain domains (CH1, CH2 and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable domain (VL), also called variable light chain domain or light chain variable region, followed by a constant light chain (CL) domain.

[0122] The term "package insert" is used to refer to instructions typically included in commercial packaging of a therapeutic product that contain information regarding the indications, usage, dosage, administration, concomitant therapy, contraindications and / or warnings for the use of such therapeutic product.

[0123] As used herein, the term "payload" refers to a therapeutic agent, which may be any naturally occurring or artificially synthesized pharma- ceutical active molecule that acts on a target (e.g., a target cell) and can be introduced into an exosome and / or producer cell. It includes therapeutic agents such as nucleotides, nucleic acids, amino acids, polypeptides, lipids, carbohydrates, viruses and viral particles, and small molecules.

[0124] "Percentage (%) amino acid sequence identity" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a reference polypeptide sequence, after aligning the sequences and introducing gaps if necessary to achieve maximum percent sequence identity, without considering any conservative substitutions as part of sequence identity for alignment. Alignment for determining percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software or FASTA program package. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the full length of the sequences being compared. Alternatively, percent identity values ​​can be generated using sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc. and the source code is on file in the user documentation of the U.S. Copyright Office, Washington DC, 20559, registered under U.S. Copyright Registration No. TXU510087, and described in WO 2001 / 007611.

[0125] Unless otherwise indicated, for purposes herein, percent amino acid sequence identity values ​​are generated using the ggsearch program of the FASTA package version 36.3.8c, or the subsequent BLOSUM50 comparison matrix. The FASTA program package was created by WR Pearson and DJ Lipman (1988), "Improved Tools for Biological Sequence Analysis", PNAS 85:2444-2448; WR Pearson (1996) "Effective protein sequence comparison" Meth. Enzymol. 266:227-258; and Pearson et.al. (1997) Genomics 46:24-36, and is publicly available at www.fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml or www.ebi.ac.uk / Tools / sss / fasta. Alternatively, sequences can be compared using the public server accessible at fasta.bioch.virginia.edu / fasta_www2 / index.cgi using the ggsearch(global protein:protein) program and default options (BLOSUM50; open:-10; ext:-2; Ktup=2) to ensure a global, rather than local, alignment. Percent amino acid identity is given in the output alignment header.

[0126] The term "pharmaceutical composition" or "pharmaceutical formulation" refers to a preparation that is in a form such that the biological activity of the active ingredients contained therein is effective and that does not contain additional ingredients that are unacceptably toxic to a subject to which the pharmaceutical composition may be administered.

[0127] A "pharmaceutically acceptable carrier" refers to an ingredient, other than an active ingredient, in a pharmaceutical composition or formulation that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, additives, stabilizers, or preservatives.

[0128] The term "TfR" or "transferrin receptor" as used herein refers to any native TfR or transferrin receptor from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses "full-length" unprocessed TfR, and any form of TfR resulting from processing within a cell. The term also encompasses naturally occurring variants of TfR, such as splice variants or allelic variants. The amino acid sequence of an exemplary human TfR is shown in SEQ ID NO:66 in Table 10.

[0129] The term "PD1" or "programmed cell death protein 1" as used herein refers to any native PD1 or programmed cell death protein 1 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses "full-length" unprocessed PD1, and any form of PD1 resulting from processing within a cell. The term also encompasses naturally occurring variants of PD1, such as splice variants or allelic variants. An exemplary amino acid sequence of human PD1 is shown in SEQ ID NO:65 in Table 10.

[0130] As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of a disease in the individual being treated, and may be performed for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, diminishing the direct or indirect pathological consequences of a disease, preventing metastasis, slowing the rate of disease progression, ameliorating or alleviating the condition, and remission, or improving prognosis. In some embodiments, the antibodies of the invention are used to delay the onset of disease or to slow the progression of a disease.

[0131] The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth. Aspects of cancer include solid tumor cancers and non-solid tumor cancers. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid tumors.More specific examples of such cancers include, but are not limited to, bladder cancer (e.g., urothelial carcinoma (UC), e.g., metastatic UC (mUC); muscle invasive bladder cancer (MIBC) and non-muscle invasive bladder cancer (NMIBC)); kidney or renal cancer (e.g., renal cell carcinoma (RCC)); lung cancer, e.g., small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung; cancer of the urinary tract, breast cancer (e.g., HER2+ breast cancer and triple negative breast cancer (TNBC), which are also known as endothelial cancers. ER-, PR- and HER2-negative; prostate cancer, e.g., castration-resistant prostate cancer (CRPC); cancer of the peritoneum; hepatocellular carcinoma; gastric cancer, or cancer of the stomach, e.g., gastrointestinal cancer and gastrointestinal stromal cancer; pancreatic cancer (e.g., pancreatic ductal adenocarcinoma (PDAC)); glioblastoma; cervical cancer; ovarian cancer; liver cancer (e.g., hepatocellular carcinoma (HCC)); hepatic cancer; colon cancer; rectal cancer; colorectal cancer; endometrial or uterine cancer; salivary gland cancer; prostate Adenocarcinoma;vulvar cancer;thyroid cancer;liver cancer;anal cancer;penile cancer;melanoma, e.g., superficial spreading melanoma, lentigo maligna melanoma, acral lentigo melanoma and nodular melanoma;multiple myeloma and B-cell lymphoma (low-grade / follicular non-Hodgkin's lymphoma (NHL);small lymphocytic (SL) NHL;intermediate-grade / follicular NHL;intermediate-grade diffuse NHL;high-grade immunoblastic NHL;high-grade lymphoblastic NHL;high-grade small noncleavable cell NHL;bulk disease NHL;mantle cell lymphoma;AID S-related lymphoma; and Waldenstrom's macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); hairy cell leukemia; chronic myeloblastic leukemia (CML); post-transplant lymphoproliferative disorder (PTLD); and myelodysplastic syndromes (MDS), as well as abnormal blood vessel proliferation associated with phakomatosis, edema (such as that associated with brain tumors), Meigs syndrome, brain cancer, head and neck cancer, and associated metastases.

[0132] As used herein, "tumor" refers to any neoplastic cell growth and proliferation, whether malignant or benign, and any pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive when referred to herein.

[0133] The terms "cell proliferative disorder" and "proliferative disorder" refer to disorders associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer. In another embodiment, the cell proliferative disorder is a tumor.

[0134] The term "B cell proliferative disorder" or "B cell malignancy" refers to disorders associated with some degree of abnormal B cell proliferation, including, for example, lymphoma, leukemia, myeloma, and myelodysplastic syndrome. In one embodiment, the B cell proliferative disorder is a lymphoma, such as non-Hodgkin's lymphoma (NHL), including, for example, DLBCL (e.g., relapsed or refractory DLBCL), FL (e.g., relapsed or refractory FL or transformed FL), or MCL. In another embodiment, the B cell proliferative disorder is a leukemia, such as chronic lymphocytic leukemia (CLL). In yet another embodiment, the B cell proliferative disorder is a central nervous system lymphoma (CNSL).

[0135] Bladder cancer The term "bladder cancer" includes, but is not limited to, urothelial carcinoma (UC), which may be, for example, locally advanced or metastatic. The methods described herein are suitable for treating various stages of cancer, including cancer that is locally advanced and / or metastatic. In cancer staging, locally advanced is generally defined as cancer that has spread from the local region to nearby tissues and / or lymph nodes. In the Roman numeral staging system, locally advanced is usually classified as stage II or III. Cancer that is metastatic is a stage (stage IV) where the cancer has spread to distant tissues and organs throughout the body.

[0136] The term "upper tract UC" refers to UC of the renal pelvis or ureter. Upper tract UC can be upper tract metastatic UC. A minority of UC cases (e.g., about 5-10%) are upper tract UC.

[0137] The term "lower urinary tract UC" refers to UC of the bladder or urethra. Lower urinary tract UC can be lower urinary tract metastatic UC. The majority of UC cases (e.g., about 90-95%) are lower urinary tract UC.

[0138] As used herein, the terms "inoperable" and "unresectable" are used interchangeably to refer to cancer (e.g., bladder cancer (e.g., UC, including locally advanced or metastatic UC)) for which surgical resection is not possible or cannot be performed safely. In some embodiments, the bladder cancer (e.g., UC, including locally advanced or metastatic UC) is inoperable due to invasion of the pelvic sidewall or adjacent viscera (clinical stage T4b) or bulky nodal metastases (N2-N3).

[0139] The term "eligible for platinum-based chemotherapy treatment" means that subject is eligible for platinum-based chemotherapy treatment according to the judgment of attending physician or according to the standardized criteria for platinum-based chemotherapy eligibility known in the art.For example, the criteria described in Galsky et al.Lancet Oncol.12(3):211-4,2011 can be used to determine whether subject is eligible for cisplatin-based chemotherapy. Galsky et al. described a consensus definition of patients with metastatic UC (mUC) in which patients who meet at least one of the following are considered unsuitable for cisplatin-based chemotherapy: (i) World Health Association (WHO) or Eastern Cooperative Oncology Group (ECOG) performance status of 2 or Karnofsky performance status of 60–70%; (ii) creatinine clearance (calculated or measured) less than 1 mL / s; (iii) National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) v4.0 grade ≥ 2 hearing loss; (iv) CTCAE v.4.0 grade ≥ 2 peripheral neuropathy; and / or New York Heart Association (NYHA) class III heart failure. In one example, a patient is considered unsuitable for cisplatin-based chemotherapy if they have one or more of the following: renal impairment (e.g., glomerular filtration rate (GFR) >30 but <60 mL / min); GFR can be assessed by direct measurement (i.e., creatinine clearance or ethyldiaminetetraacetate) or, if not available, by calculation from serum / plasma creatinine (Cockcroft-Gault formula); hearing loss (e.g., National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) v4.0 grade ≥2, hearing loss of 25 decibels at two consecutive frequencies); peripheral neuropathy (e.g., NCI CTCAE v4.0 grade ≥2 peripheral neuropathy (i.e., sensory changes or paresthesias, including tingling)); and / or ECOG performance status assessment (see Oken et al. Am. J. Clin. Oncol. 5:649-655, 1982) (e.g., ECOG performance status of 2).In some embodiments, subjects may be eligible for carboplatin-based chemotherapy if they have one of the following: renal impairment (e.g., glomerular filtration rate (GFR) >30 but <60 mL / min); GFR may be assessed by direct measurement (i.e., creatinine clearance or ethyldiaminetetraacetate) or, if not available, by calculation from serum / plasma creatinine (Cockcroft-Gault formula); hearing loss (e.g., CTCAE v4.0 grade ≧2 hearing loss of 25 decibels at two consecutive frequencies); peripheral neuropathy (e.g., NCI CTCAE v4.0 grade ≧2 peripheral neuropathy (i.e., sensory changes or paresthesias including tingling)); and / or ECOG performance status assessment (e.g., ECOG performance status of 2).

[0140] Chemotherapeutic agents also include "platinum-based" chemotherapeutic agents, which include organic compounds that contain platinum as an integral part of the molecule. Typically, platinum-based chemotherapeutic agents are platinum coordination complexes. Platinum-based chemotherapeutic agents are sometimes referred to in the art as "platins". Examples of platinum-based chemotherapeutic agents include, but are not limited to, cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, lipoplatin, and satraplatin. Platinum-based chemotherapeutic agents (e.g., cisplatin or carboplatin) can be administered in combination with one or more additional chemotherapeutic agents, such as nucleoside analogs (e.g., gemcitabine).

[0141] As used herein, "platinum-based chemotherapy" refers to a chemotherapy regimen that includes a platinum-based chemotherapy agent.For example, platinum-based chemotherapy can include a platinum-based chemotherapy agent (e.g., cisplatin or carboplatin) in combination with one or more additional chemotherapy agents, such as, for example, a nucleoside analog (e.g., gemcitabine).

[0142] As used herein, "nucleoside analog" refers to a nucleoside that comprises a nucleic acid analog and a sugar. Nucleoside analogs can function as antimetabolites. Exemplary nucleoside analogs include, but are not limited to, gemcitabine, cytarabine, fludarabine and cladribine.

[0143] Breast cancer The term "breast cancer" includes, but is not limited to, HER2+ breast cancer, as well as triple-negative breast cancer (TNBC), which is a form of breast cancer in which the cancer cells are negative for estrogen receptors (ER-), progesterone receptors (PR-), and HER2 (HER2-), and may be locally advanced, unresectable, and / or metastatic (e.g., metastatic triple-negative breast cancer (mTNBC)).

[0144] As used herein, the terms "early TNBC" and "eTNBC" refer to early TNBC, including stages I-III TNBC. Early TNBC accounts for 10%-20% of all new early breast cancer diagnoses and has a 3-year event-free survival rate of 74%-76% after treatment with neoadjuvant anthracycline and taxane therapy.

[0145] As used herein, "pathological complete response" or "pCR" refers to the disappearance of invasive tumor from both the breast and lymph nodes. The term pCR includes the absence of invasive cancer in the breast and axillary lymph nodes regardless of ductal carcinoma in situ (i.e., ypT0 / is ypN0); the absence of invasive cancer and carcinoma in situ in the breast and axillary lymph nodes (i.e., ypT0 ypN0); and the absence of invasive cancer in the breast regardless of ductal carcinoma in situ or lymph node metastasis (i.e., ypT0 / is). In certain embodiments, pCR refers to the absence of invasive cancer in the breast and axillary lymph nodes regardless of ductal carcinoma in situ (i.e., ypT0 / is ypN0).

[0146] As used herein, a "taxane" is an agent (e.g., a diterpene) that can bind to tubulin, promote microtubule assembly and stabilization, and / or prevent microtubule depolymerization. Exemplary taxanes include, but are not limited to, paclitaxel (i.e., TAXOL®, CAS#33069-62-4), docetaxel (i.e., TAXOTERE®, CAS#114977-28-5), larotaxel, cabazitaxel, mirataxel, tesetaxel, and / or orataxel. Taxanes included herein also include the taxoid 10-deacetylbaccatin III and / or derivatives thereof. In some embodiments, the taxane is an albumin-coated nanoparticle (e.g., nano-albumin-bound (nab)-paclitaxel, i.e., ABRAXANE®, and / or nab-docetaxel, ABI-008). In some embodiments, the taxane is nab-paclitaxel (ABRAXANE®). In some embodiments, the taxane is formulated in CREMAPHOR® (e.g., TAXOL®) and / or TWEEN®, e.g., polysorbate 80 (e.g., TAXOTERE®). In some embodiments, the taxane is liposomally encapsulated taxane. In some embodiments, the taxane is a prodrug and / or conjugated form of the taxane (e.g., paclitaxel, paclitaxel poliglumex, and / or DHA covalently conjugated to linoleyl carbonate-paclitaxel). In some embodiments, the paclitaxel is formulated substantially without surfactants (e.g., in the absence of CREMAPHOR® and / or TWEEN®, e.g., TOCOSOL® paclitaxel).

[0147] "Anthracycline" as used herein refers to a class of antibiotic compounds that exhibit cytotoxic activity. Anthracyclines can cause cytotoxicity through DNA intercalation, topoisomerase II-mediated toxicity, generation of reactive oxygen species, and / or DNA adduct formation. Exemplary anthracyclines include, but are not limited to, doxorubicin, epirubicin, idarubicin, daunorubicin, mitoxantrone, and valrubicin. In some embodiments, the anthracycline is doxorubicin or epirubicin. In some specific embodiments, the anthracycline is doxorubicin. In other specific embodiments, the anthracycline is epirubicin.

[0148] As used herein, "alkylating agent" refers to a class of chemotherapeutic agents that attach alkyl groups to nucleotides, e.g., DNA. Typically, the alkyl group is attached to a guanine base of DNA. Exemplary alkylating agents include, but are not limited to, nitrogen mustard derivatives (e.g., cyclophosphamide, chlorambucil, uramustine, melphalan, or bendamustine), nitrosoureas (e.g., carmustine, lomustine, or streptozocin), alkylsulfonates (e.g., busulfan), triazines (e.g., dacarbazine or temozolomide), and ethylenimines (e.g., altretamine or thiotepa).

[0149] Kidney cancer In some embodiments, the cancer is renal cancer. In certain embodiments, the renal cancer is renal cell carcinoma (RCC) (e.g., advanced RCC or metastatic RCC (mRCC), including previously untreated RCC). In some embodiments, the renal cancer is sarcomatoid renal cancer (e.g., sarcomatoid RCC (e.g., sarcomatoid advanced or mRCC)).

[0150] The term "sarcomatoid" refers to a cancer (e.g., a kidney cancer (e.g., RCC)) that is characterized by a sarcomatoid morphology, for example, as assessed by histology. Sarcomatoid kidney cancer (e.g., sarcomatoid RCC) is associated with aggressive behavior and poor prognosis. In some embodiments, sarcomatoid kidney cancer comprises or consists of atypical spindle cells and / or resembles the morphology of any sarcoma. See, for example, El Mouallem et al. Urol. Oncol. 36:265-271, 2018, which is incorporated herein by reference in its entirety. Sarcomatoid RCC can occur in any subtype of RCC, including clear cell RCC, chromophobe RCC, collecting duct carcinoma, renal medullary carcinoma, fumarate hydratase (FH)-deficient RCC, and succinate dehydrogenase (SDH)-deficient RCC. The incidence of sarcomatoid RCC varies by subtype but is usually higher in clear cell RCC (approximately 5-8%) and chromophobe RCC (approximately 8-10%). The histology of the sarcomatoid component can be variable and may include a fibrosarcomatoid pattern, a pleomorphic undifferentiated sarcomatoid pattern or other heterologous sarcomatoid patterns (e.g., osteosarcomatoid, chondrosarcomatoid or rhabdomyosarcomatoid patterns). Necrosis is typically present in the majority (approximately 90%) of cases. In some embodiments, there is no minimum amount or percentage of sarcomatoid differentiation for an individual's kidney cancer to be classified as sarcomatoid. Sarcomatoid RCC may be evaluated as described in Example 1. In other embodiments, sarcomatoid RCC may be characterized as described by the 2012 International Society of Urological Pathology (ISUP) Vancouver Consensus (see Srigley et al. Am. J. Surg. Pathol. 37:1469-89, 2013, incorporated herein by reference in its entirety).

[0151] The term "Memorial Sloan Kettering Cancer Center (MSKCC) risk score" refers to a scoring system based on a set of prognostic factors related to the survival of kidney cancer (e.g., RCC, e.g., mRCC) patients.See, for example, Motzer et al. J.Clin.Oncol.17(8):2530-2540,1999 and Motzer et al. J.Clin.Oncol.20(1):289-296,2002, the entire contents of which are incorporated herein by reference. In some embodiments, the MSKCC risk score may be calculated based on the following factors, as described in Example 1: (i) time from nephrectomy to treatment (e.g., systemic treatment) less than 1 year, lack of nephrectomy, or initial diagnosis of metastatic disease; (ii) hemoglobin level below the lower limit of normal (LLN) (in some cases, the normal range for hemoglobin is 13.5-17.5 g / dL for men and 12-15.5 g / dL for women); (iii) corrected serum calcium level greater than 10 mg / dL (in some cases, the corrected serum calcium level is serum calcium level (mg / dL) + 0.8(4-serum albumin (g / dL))); (iv) serum lactate dehydrogenase (LDH) level greater than 1.5 times the upper limit of normal (ULN) (in some cases, the ULN is 140 U / L); and / or (v) Karnofsky Performance Status (KPS) score less than 80. In some embodiments, if an individual does not have the above characteristics, the individual has a favorable MSKCC risk score. In some embodiments, if an individual has one or two of the above characteristics, the individual has a moderate MSKCC risk score. In some embodiments, if an individual has three or more of the above characteristics, the individual has a poor MSKCC risk score. In some embodiments, an individual's MSKCC risk score can be used to identify whether the individual may benefit from anti-cancer treatment, for example, anti-cancer treatment including VEGF antagonists (e.g., anti-VEGF antibodies such as bevacizumab) and PD-L1 axis binding antagonists (e.g., anti-PD-L1 antibodies such as atezolizumab).

[0152] As used herein, "deterioration-free rate" or "DFR" refers to the probability that a patient will experience a clinically meaningful worsening over a given period of time, e.g., the length of time from the start of treatment to the patient's first 2 or more point increase above baseline on the MD Anderson Symptom Assessment Inventory (MDASI) Interference scale.

[0153] The "MD Anderson Symptom Assessment Inventory (MDASI) Interference scale" refers to a patient-reported outcome measurement scoring system that assesses the severity and impact of multiple symptoms related to cancer and its treatment (see, e.g., Mendoza et al. Clin. Breast Cancer 13:325-334, 2013; Jones et al. Clin. Genitourin. Cancer 12:41-49, 2014; and Shi et al. Pain 158:1108-1112, 2017). In the MDASI Interference scale, patients rate how much their symptoms have interfered with various aspects of their life during the past 24 hours. Each interference item (work, general activities, walking, relationships with others, enjoyment of life, and mood) is rated on a scale of 0 to 10, with 0 representing "not interfering" and 10 representing "completely interfering."

[0154] Liver cancer As used herein, the term "unresectable" refers to a cancer for which surgical resection is not possible or cannot be performed safely (e.g., liver cancer (e.g., HCC, including locally advanced or metastatic and / or unresectable HCC)). For example, with respect to liver cancer, the term "unresectable" indicates that the cancer cannot be safely removed by partial hepatectomy, for example, because the tumor is too large to be safely removed, the tumor is located in a part of the liver that makes removal difficult (e.g., near a large blood vessel), there are several tumors, or the cancer has spread throughout the liver and / or outside the liver, and / or the subject has an underlying health problem (e.g., cirrhosis) that makes resection impossible.

[0155] As used herein, "time to radiographic progression" (TTRP) refers to the length of time between a first event (e.g., randomization into a clinical trial or administration of the first dose of a treatment regimen) and objective progression as assessed by radiology. In some embodiments, radiographic progression is defined according to Response Evaluation Criteria in Solid Tumors (RECIST) criteria, e.g., RECIST v1.1 or mRECIST (e.g., HCC mRECIST).

[0156] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding of the antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of a natural antibody generally have a similar structure, and each domain contains four conserved framework regions (FR) and three complementarity determining regions (CDR). (See, for example, Kindt et al. Kuby Immunology, 6 th ed., WH Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen may be isolated using a VH or VL domain from an antibody that binds to that antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0157] The term "vector" as used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures and vectors integrated into the genome of a host cell into which it is introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0158] II. Compositions and Methods In one embodiment, the present invention is based in part on the discovery that combining a first antigen-binding domain that specifically binds to TfR on the one hand and a second and optionally a third antigen-binding domain that specifically binds to PD1 on the other hand in a single bispecific antibody results in internalization of the bispecific antibody when contacted with a cell that expresses and / or displays TfR and PD1 on its surface. Such a cell may be, for example, an activated T cell. In a further embodiment, the present invention is based in part on the discovery that anti-TfR anti-PD1 2+1 format antibodies, i.e., bispecific antibodies comprising a first antigen-binding domain that specifically binds to TfR and a second and a third antigen-binding domain that specifically bind to PD1, exhibit improved biological activity over monospecific bivalent PD1 antibodies and result in better inhibition of the interaction between PD1 and PD-L1. In one embodiment, these molecules also comprise an IgG class Fab fragment, which are covalently linked to each other to result in the 2+1 format antibody disclosed herein, and optionally an IgG class Fc region. Without wishing to be bound by theory, the inventors believe that these antibodies deplete PD1 from the surface of T cells, thus permanently preventing PD1 binding to PD1 ligand (PD-L1) located on the surface of tumor cells, more than can be achieved by the simple binding of anti-PD1 blocking antibodies to PD1. Thus, the antibodies of the present invention provide a stronger inhibitory effect on PD1 / PD-L1-mediated signaling than anti-PD1 antibodies known in the art. The usefulness of the antibodies described herein is believed to be related to their ability to form a complex with two receptors, TfR and PD1, on the cell surface, thereby internalizing the entire complex into the cell.

[0159] The antibody according to the present invention can also be used to specifically deliver payload to T cells by directly conjugating the payload to the antibody or by attaching an additional antigen-binding domain that binds to the antibody, and then the antibody can specifically bind to the payload.In certain embodiments, a bispecific antibody is provided that binds to PD1 and TfR.The antibody of the present invention is useful for delivering agents such as small molecules or RNA to T cells for cancer treatment, for example.

[0160] In another embodiment, the present invention is based in part on the discovery that bispecific anti-TfR anti-PD1 antibodies with a stoichiometry of 2+1 between anti-PD1 and anti-TfR targeting binding domains, i.e., bispecific antibodies comprising a first antigen-binding domain that specifically binds TfR and a second and third antigen-binding domain that specifically binds PD1, result in increased internalization of PD1 compared to monospecific anti-PD1 antibodies with a stoichiometry of 1+1 between anti-PD1 and anti-TfR targeting binding domains or bispecific anti-TfR anti-PD1 antibodies, i.e., bispecific antibodies comprising one antigen-binding domain that specifically binds TfR and only one antigen-binding domain that specifically binds PD1. The antibodies of the present invention are useful, for example, for inhibiting the growth of tumor cells in an individual or for the diagnosis or treatment of tumors. The antibodies of the present invention can be used in any type of medical treatment in which anti-PD1 antibodies known in the art are used.

[0161] A. Exemplary Bispecific Antibodies that Bind TfR and PD1 In one embodiment, the present invention provides a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second and optionally a third antigen-binding domain that specifically binds to PD1. In one embodiment, an isolated bispecific antibody is provided comprising a first antigen-binding domain that specifically binds to TfR and a second and optionally a third antigen-binding domain that specifically binds to PD1. In one embodiment, the present invention provides an antibody that specifically binds to TfR and PD1. In a particular embodiment, a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second and optionally a third antigen-binding domain that specifically binds to PD1 causes depletion of PD1 from the cell surface and causes internalization of PD1 into the cell. The cell is preferably a T cell, more preferably an activated T cell. Internalization of PD1 into the cell inhibits binding of PD1 to its ligand, preferably binding to PD-L1. Preferably, this inhibition of binding between PD1 and its ligands by internalization of PD1 into the cell has a longer lasting effect than the block achieved by mere binding of the antigen-binding domain to PD1. A bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second and optionally a third antigen-binding domain that specifically binds to PD1 is preferably i. Binds to TfR and PD1 simultaneously ii. binding simultaneously to TfR and PD1, resulting in internalization of the complex formed by the bispecific antibody, TfR and PD1 into the cell and depletion of PD1 from the cell surface, preventing PD-L1 from accessing PD1; and / or iii. can block PD1-PD-L1 interaction and / or prevent PD1 signaling by binding to PD1 and achieving depletion of PD1 from the cell surface, preferably resulting in a more effective and / or permanent inhibition of PD1 / PD-L1 mediated signaling than conventional anti-PD1 antibodies.

[0162] In one embodiment, the Fab fragments and optionally also the IgG class Fc region contained in the bispecific antibody are covalently linked to each other, resulting in a 2+1 format antibody of different conformation. In a further embodiment, the bispecific antibody is essentially in monomeric form, i.e., does not form a dimeric or multimeric (e.g., pentameric) structure comprising multiple bispecific antibodies of the invention. In a particular embodiment, at least 90%, more particularly at least 95%, preferably at least 98%, and more preferably at least 99% of the antibody is in monomeric form.

[0163] In one aspect, the present invention provides a method for producing a method for treating a cancer cell comprising: A) a. at least one, at least two, at least three, at least four, at least five, or all six CDRs selected from: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6; or b. At least one, at least two, at least three, at least four, at least five, or all six CDRs selected from: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:9; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:10; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:11; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:12; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:13; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:14. A first antigen-binding domain that specifically binds to TfR, comprising: B) a. at least one, at least two, at least three, at least four, at least five, or all six CDRs selected from: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 17; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 18; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 19; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 20; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 21; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 22; or b. At least one, at least two, at least three, at least four, at least five, or all six CDRs selected from: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 25; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 26; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 27; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 28; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 29; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 30. and a second, and optionally a third, antigen-binding domain that specifically binds to PD1, comprising:

[0164] In one aspect, the present invention provides a method for producing a method for treating a cancer cell comprising: A) at least one, at least two, or all three VH CDR sequences selected from: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:1; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:2; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:3; or b. At least one, at least two, or all three VH CDR sequences selected from: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:9; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:10; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:11. A first antigen-binding domain that specifically binds to TfR, comprising: B) a. at least one, at least two, or all three VH CDR sequences selected from: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 17; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 18; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 19; or b. At least one, at least two, or all three VH CDR sequences selected from: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:25; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:26; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:27. and a second, and optionally a third, antigen-binding domain that specifically binds to PD1, comprising:

[0165] In one embodiment, the bispecific antibody comprises a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3 and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 19. In one embodiment, the bispecific antibody comprises a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3 and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 27.

[0166] In one embodiment, the bispecific antibody comprises a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11 and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 19. In one embodiment, the bispecific antibody comprises a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11 and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 27.

[0167] In another aspect, the bispecific antibody comprises: A) a. a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6, or b. CDR-H3 comprising the amino acid sequence of SEQ ID NO:11, and CDR-L3 comprising the amino acid sequence of SEQ ID NO:14 A first antigen-binding domain that specifically binds to TfR, comprising: B) a. a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 19, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 22; or b. CDR-H3 comprising the amino acid sequence of SEQ ID NO:27, and CDR-L3 comprising the amino acid sequence of SEQ ID NO:30 and optionally a third antigen-binding domain that specifically binds to PD1.

[0168] In a further aspect, the antibody comprises: A) a. CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6, and CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; or b. CDR-H3 comprising the amino acid sequence of SEQ ID NO:11, CDR-L3 comprising the amino acid sequence of SEQ ID NO:14, and CDR-H2 comprising the amino acid sequence of SEQ ID NO:10 A first antigen-binding domain that specifically binds to TfR, comprising: And B) a. CDR-H3 comprising the amino acid sequence of SEQ ID NO: 19, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 22, and CDR-H2 comprising the amino acid sequence of SEQ ID NO: 18; or b. CDR-H3 comprising the amino acid sequence of SEQ ID NO:27, CDR-L3 comprising the amino acid sequence of SEQ ID NO:30, and CDR-H2 comprising the amino acid sequence of SEQ ID NO:26 and optionally a third antigen-binding domain that specifically binds to PD1.

[0169] In a further aspect, the antibody comprises: A) a. CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; or b. CDR-H1 comprising the amino acid sequence of SEQ ID NO:9; CDR-H2 comprising the amino acid sequence of SEQ ID NO:10; and CDR-H3 comprising the amino acid sequence of SEQ ID NO:11. A first antigen-binding domain that specifically binds to TfR, comprising: And B) a. CDR-H1 comprising the amino acid sequence of SEQ ID NO: 17; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 18; and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 19; or b. CDR-H1 comprising the amino acid sequence of SEQ ID NO:25; CDR-H2 comprising the amino acid sequence of SEQ ID NO:26; and CDR-H3 comprising the amino acid sequence of SEQ ID NO:27. and optionally a third antigen-binding domain that specifically binds to PD1.

[0170] In another aspect, the present invention provides a method for producing a composition comprising: A) at least one, at least two, or all three VL CDR sequences selected from: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO:4; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO:5; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO:6; or b. At least one, at least two, or all three VL CDR sequences selected from: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 14. A first antigen-binding domain that specifically binds to TfR, comprising: B) a. at least one, at least two, or all three VL CDR sequences selected from: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 20; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 21; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 22; or b. At least one, at least two, or all three VL CDR sequences selected from: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO:28; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO:29; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO:30. and a second, and optionally a third, antigen-binding domain that specifically binds to PD1, comprising:

[0171] In one aspect, the bispecific antibody comprises: A) a. (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6, or b. (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 14. A first antigen-binding domain that specifically binds to TfR, comprising: B) a. (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 20; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 21; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 22, or b. (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO:28; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO:29; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO:30. and optionally a third antigen-binding domain that specifically binds to PD1.

[0172] In another aspect, the bispecific antibody of the invention comprises: A) a. a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6. or b. A VH domain comprising at least one, at least two, or all three VH CDR sequences selected from (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11, and a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 12, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 14. A first antigen-binding domain that specifically binds to TfR, comprising: B) a. a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 17, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 18, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 19, and a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 20, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 21, and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 22. or b. A VH domain comprising at least one, at least two, or all three VH CDR sequences selected from (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:25, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:26, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO:27, and a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO:28, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO:30. and optionally a third antigen-binding domain that specifically binds to PD1.

[0173] In another aspect, the present invention provides a method for producing a composition comprising: A) a first antigen-binding domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6; B) A bispecific antibody comprising a second and, optionally, a third antigen-binding domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 17; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 18; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 19; (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 20; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 21; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 22.

[0174] In another aspect, the present invention provides a method for producing a composition comprising: C) a first antigen-binding domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6. D) a second and, optionally, a third antigen-binding domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 25; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 26; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 27; (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 28; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 29; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 30.

[0175] In another aspect, the present invention provides a method for producing a composition comprising: E) a first antigen-binding domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:9; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:10; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:11; (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:12; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:13; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:14. F) A bispecific antibody comprising a second and, optionally, a third antigen-binding domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 17; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 18; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 19; (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 20; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 21; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 22.

[0176] In another aspect, the present invention provides a method for producing a composition comprising: G) a first antigen-binding domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:9; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:10; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:11; (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:12; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:13; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:14. H) a second, and optionally a third, antigen-binding domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 25; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 26; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 27; (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 28; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 29; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 30.

[0177] In any of the aspects provided herein, a bispecific antibody comprising a first antigen-binding domain that specifically binds TfR and a second and optionally a third antigen-binding domain that specifically binds PD1 is humanized. In one aspect, the anti-TfR anti-PD1 bispecific antibody further comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework. The acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence or may contain amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0178] In another aspect, a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second and optionally a third antigen-binding domain that specifically binds to PD1 comprises one or more of the VH CDR sequences of SEQ ID NO: 7 and one or more of the VH CDR sequences of SEQ ID NO: 23. In another embodiment, a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second and optionally a third antigen-binding domain that specifically binds to PD1 comprises one or more of the VL CDR sequences of SEQ ID NO: 8 and one or more of the VL CDR sequences of SEQ ID NO: 24. In another embodiment, a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second and optionally a third antigen-binding domain that specifically binds to PD1 comprises the VH CDR sequence of SEQ ID NO: 7, and the VL CDR sequence of SEQ ID NO: 8, and the VH CDR sequence of SEQ ID NO: 23, and the VL CDR sequence of SEQ ID NO: 24.

[0179] In another aspect, a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second and optionally a third antigen-binding domain that specifically binds to PD1 comprises one or more of the VH CDR sequences of SEQ ID NO: 7 and one or more of the VH CDR sequences of SEQ ID NO: 31. In another embodiment, a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second and optionally a third antigen-binding domain that specifically binds to PD1 comprises one or more of the VL CDR sequences of SEQ ID NO: 8 and one or more of the VL CDR sequences of SEQ ID NO: 32. In another embodiment, a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second and optionally a third antigen-binding domain that specifically binds to PD1 comprises the VH CDR sequence of SEQ ID NO: 7, and the VL CDR sequence of SEQ ID NO: 8, and the VH CDR sequence of SEQ ID NO: 31, and the VL CDR sequence of SEQ ID NO: 32.

[0180] In another aspect, a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second and optionally a third antigen-binding domain that specifically binds to PD1 comprises one or more of the VH CDR sequences of SEQ ID NO: 15 and one or more of the VH CDR sequences of SEQ ID NO: 23. In another embodiment, a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second and optionally a third antigen-binding domain that specifically binds to PD1 comprises one or more of the VL CDR sequences of SEQ ID NO: 16 and one or more of the VL CDR sequences of SEQ ID NO: 24. In another embodiment, a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second and optionally a third antigen-binding domain that specifically binds to PD1 comprises the VH CDR sequence of SEQ ID NO: 15, and the VL CDR sequence of SEQ ID NO: 16, and the VH CDR sequence of SEQ ID NO: 23, and the VL CDR sequence of SEQ ID NO: 24.

[0181] In another aspect, a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second and optionally a third antigen-binding domain that specifically binds to PD1 comprises one or more of the VH CDR sequences of SEQ ID NO: 15 and one or more of the VH CDR sequences of SEQ ID NO: 31. In another embodiment, a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second and optionally a third antigen-binding domain that specifically binds to PD1 comprises one or more of the VL CDR sequences of SEQ ID NO: 16 and one or more of the VL CDR sequences of SEQ ID NO: 32. In another embodiment, a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second and optionally a third antigen-binding domain that specifically binds to PD1 comprises the VH CDR sequence of SEQ ID NO: 15, and the VL CDR sequence of SEQ ID NO: 16, and the VH CDR sequence of SEQ ID NO: 31, and the VL CDR sequence of SEQ ID NO: 32.

[0182] In a further embodiment, a bispecific antibody comprising a first antigen-binding domain that specifically binds TfR and a second and optionally a third antigen-binding domain that specifically binds PD1 comprises the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of the VH domain of SEQ ID NO: 7, and the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of the VL domain of SEQ ID NO: 8, and the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of the VH domain of SEQ ID NO: 23, and the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of the VL domain of SEQ ID NO: 24.

[0183] In a further embodiment, a bispecific antibody comprising a first antigen-binding domain that specifically binds TfR and a second and optionally a third antigen-binding domain that specifically binds PD1 comprises the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of the VH domain of SEQ ID NO: 7, and the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of the VL domain of SEQ ID NO: 8, and the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of the VH domain of SEQ ID NO: 31, and the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of the VL domain of SEQ ID NO: 32.

[0184] In a further embodiment, a bispecific antibody comprising a first antigen-binding domain that specifically binds TfR and a second and optionally a third antigen-binding domain that specifically binds PD1 comprises the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of the VH domain of SEQ ID NO: 15, and the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of the VL domain of SEQ ID NO: 16, and the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of the VH domain of SEQ ID NO: 23, and the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of the VL domain of SEQ ID NO: 24.

[0185] In a further embodiment, a bispecific antibody comprising a first antigen-binding domain that specifically binds TfR and a second and optionally a third antigen-binding domain that specifically binds PD1 comprises the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of the VH domain of SEQ ID NO: 15, and the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of the VL domain of SEQ ID NO: 16, and the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of the VH domain of SEQ ID NO: 31, and the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of the VL domain of SEQ ID NO: 32.

[0186] In one embodiment, a bispecific antibody comprising a first antigen-binding domain which specifically binds to TfR and a second and optionally a third antigen-binding domain which specifically binds to PD1 comprises a) one or more of the heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 7 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequences of the VH domain of SEQ ID NO: 7; and b) one or more of the heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 23 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequences of the VH domain of SEQ ID NO: 23.

[0187] In one embodiment a bispecific antibody comprising a first antigen-binding domain which specifically binds to TfR and a second and optionally a third antigen-binding domain which specifically binds to a PD1 antibody comprises a) the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 7 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequences of the VH domain of SEQ ID NO: 7; and b) the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 23 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequences of the VH domain of SEQ ID NO: 23.

[0188] In one embodiment a bispecific antibody comprising a first antigen-binding domain which specifically binds to TfR and a second and optionally a third antigen-binding domain which specifically binds to PD1 comprises a) the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 7 and a framework of at least 95% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 7, and b) the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 23 and a framework of at least 95% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 23. In another embodiment, a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second and optionally a third antigen-binding domain that specifically binds to PD1 comprises a) the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 7 and a framework with at least 98% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 23, and b) the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 7 and a framework with at least 98% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 23.

[0189] In one embodiment, a bispecific antibody comprising a first antigen-binding domain which specifically binds to TfR and a second and optionally a third antigen-binding domain which specifically binds to PD1 comprises a) one or more of the heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 7 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequences of the VH domain of SEQ ID NO: 7; and b) one or more of the heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 31 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequences of the VH domain of SEQ ID NO: 31.

[0190] In one embodiment, a bispecific antibody comprising a first antigen-binding domain which specifically binds to TfR and a second and optionally a third antigen-binding domain which specifically binds to a PD1 antibody comprises a) the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 7 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 7, and b) the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 31 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 31. In one embodiment a bispecific antibody comprising a first antigen-binding domain which specifically binds to TfR and a second and optionally a third antigen-binding domain which specifically binds to PD1 comprises a) the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 7 and a framework of at least 95% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 7, and b) the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 31 and a framework of at least 95% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 31. In another embodiment, a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second and optionally a third antigen-binding domain that specifically binds to PD1 comprises a) the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 7 and a framework with at least 98% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 7, and b) the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 31 and a framework with at least 98% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 31.

[0191] In one embodiment, a bispecific antibody comprising a first antigen-binding domain which specifically binds to TfR and a second and optionally a third antigen-binding domain which specifically binds to PD1 comprises a) one or more of the heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 15 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequences of the VH domain of SEQ ID NO: 15; and b) one or more of the heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 23 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequences of the VH domain of SEQ ID NO: 23.

[0192] In one embodiment a bispecific antibody comprising a first antigen-binding domain which specifically binds to TfR and a second and optionally a third antigen-binding domain which specifically binds to a PD1 antibody comprises a) the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 15 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 15; and b) the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 23 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 23. In one embodiment a bispecific antibody comprising a first antigen-binding domain which specifically binds to TfR and a second and optionally a third antigen-binding domain which specifically binds to PD1 comprises a) the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 15 and a framework of at least 95% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 15, and b) the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 23 and a framework of at least 95% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 23. In another embodiment, a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second and optionally a third antigen-binding domain that specifically binds to PD1 comprises a) the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 15 and a framework of at least 98% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 15, and b) the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 23 and a framework of at least 98% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 23.

[0193] In one embodiment, a bispecific antibody comprising a first antigen-binding domain which specifically binds to TfR and a second and optionally a third antigen-binding domain which specifically binds to PD1 comprises a) one or more of the heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 15 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequences of the VH domain of SEQ ID NO: 15; and b) one or more of the heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 31 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequences of the VH domain of SEQ ID NO: 31.

[0194] In one embodiment a bispecific antibody comprising a first antigen-binding domain which specifically binds to TfR and a second and optionally a third antigen-binding domain which specifically binds to PD1 comprises a) the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 15 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 15; and b) the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 31 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 31. In one embodiment a bispecific antibody comprising a first antigen-binding domain which specifically binds to TfR and a second and optionally a third antigen-binding domain which specifically binds to PD1 comprises a) the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 15 and a framework of at least 95% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 15, and b) the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 31 and a framework of at least 95% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 31. In another embodiment, a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second and optionally a third antigen-binding domain that specifically binds to PD1 comprises a) the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 15 and a framework of at least 98% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 15, and b) the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 31 and a framework of at least 98% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 31.

[0195] In one embodiment a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second and optionally a third antigen-binding domain that specifically binds to PD1 comprises A) a first antigen-binding domain that specifically binds to TfR, comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 7 and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 8; B) (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 17; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 18; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 19; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 20; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 21; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 22, and a CDR-L4 comprising at least 90%, 91%, 92%, 100%, 150 ...50%, 100%, 150%, 150%, 100%, 150%, 150%, 150%, 150%, 150%, 150%, 15 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:24; and a second antigen-binding domain and / or, if present, a third antigen-binding domain that specifically binds PD1, comprising a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:24.

[0196] In one aspect, the first VH domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 7 and the second VH domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 23. In one aspect, the first VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 8 and the second VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 24.

[0197] In one embodiment, a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second antigen-binding domain that specifically binds to PD1 comprises: A) a first antigen-binding domain that specifically binds to TfR, comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 7 and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 8; B) (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 25; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 26; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 27; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 28; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 29; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 30, and a CDR-L4 comprising at least 90%, 91%, 92%, 100%, 150 ...50%, 100%, 150%, 150%, 150%, 150%, 150%, 150%, 150%, 150%, 150%, 150%, 150%, 150%, 150%, 150%, 150%, 150%, 150%, 150%, 150%, 15 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:32; and a second antigen-binding domain and / or, if present, a third antigen-binding domain that specifically binds PD1, comprising a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:32.

[0198] In one aspect, the first VH domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 7 and the second VH domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 31. In one aspect, the first VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 8 and the second VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 32.

[0199] In one embodiment a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second and optionally a third antigen-binding domain that specifically binds to PD1 comprises (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11; (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 14, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 15 and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 16; B) (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 17; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 18; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 19; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 20; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 21; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 22, and a CDR-L4 comprising at least 90%, 91%, 92%, 100%, 150 ...50%, 100%, 150%, 150%, 100%, 150%, 150%, 150%, 150%, 150%, 150%, 15 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:24; and a second antigen-binding domain and / or, if present, a third antigen-binding domain that specifically binds PD1, comprising a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:24.

[0200] In one aspect, the first VH domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 15 and the second VH domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 23. In one aspect, the first VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 16 and the second VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 24.

[0201] In one embodiment a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second and optionally a third antigen-binding domain that specifically binds to PD1 comprises (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11; (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 14, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 15 and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 16; B) (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 25; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 26; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 27; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 28; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 29; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 30, and a CDR-L4 comprising at least 90%, 91%, 92%, 100%, 150 ...50%, 100%, 150%, 150%, 150%, 150%, 150%, 150%, 150%, 150%, 150%, 150%, 150%, 150%, 150%, 150%, 150%, 150%, 150%, 150%, 150%, 15 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:32; and a second antigen-binding domain and / or, if present, a third antigen-binding domain that specifically binds PD1, comprising a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:32.

[0202] In one aspect, the first VH domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 15 and the second VH domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 31. In one aspect, the first VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 16 and the second VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 32.

[0203] In another embodiment, a bispecific antibody comprising a first antigen-binding domain that specifically binds TfR and a second and optionally a third antigen-binding domain that specifically binds PD1 comprises: A) a first heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 7 and SEQ ID NO: 15; B) a second heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:23 and SEQ ID NO:31.

[0204] In one embodiment a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second and optionally a third antigen-binding domain that specifically binds to PD1 comprises A) a heavy chain variable domain (VH) sequence having at least 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 7 and SEQ ID NO: 15; B) a heavy chain variable domain (VH) sequence having at least 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:23 and SEQ ID NO:31.

[0205] In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity contains substitutions (e.g., conservative substitutions), insertions or deletions compared to a reference sequence, but a bispecific antibody comprising a first antigen-binding domain that specifically binds TfR and a second and optionally a third antigen-binding domain that specifically binds PD1, comprising that sequence, retains the ability to bind TfR and / or PD1. In certain embodiments, a total of 1-10 amino acids are substituted, inserted and / or deleted in SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:23 and / or SEQ ID NO:31. In certain embodiments, the substitutions, insertions or deletions occur within the regions outside the CDRs (i.e., FRs). Optionally, a bispecific antibody comprising a first antigen-binding domain that specifically binds TfR and a second and optionally a third antigen-binding domain that specifically binds PD1 comprises a first heavy chain variable domain (VH) sequence selected from the group consisting of SEQ ID NO:7 and SEQ ID NO:15, including post-translational modifications of that sequence, and a second heavy chain variable domain (VH) sequence selected from the group consisting of SEQ ID NO:23 and SEQ ID NO:31.

[0206] In another embodiment, a bispecific antibody comprising a first antigen-binding domain that specifically binds TfR and a second and optionally a third antigen-binding domain that specifically binds PD1 comprises: A) a first light chain variable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:8 and SEQ ID NO:16; B) a second light chain variable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:24 and SEQ ID NO:32.

[0207] In one embodiment a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second and optionally a third antigen-binding domain that specifically binds to PD1 comprises A) a light chain variable domain (VL) sequence having at least 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 8 and SEQ ID NO: 16; B) a light chain variable domain (VL) sequence having at least 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:24 and SEQ ID NO:32.

[0208] In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity contains substitutions (e.g., conservative substitutions), insertions or deletions compared to a reference sequence, but a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second and optionally a third antigen-binding domain that specifically binds to PD1, comprising the sequence, retains the ability to bind to TfR and / or PD1. In certain embodiments, a total of 1-10 amino acids are substituted, inserted and / or deleted in SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:24 and / or SEQ ID NO:32. In certain embodiments, the substitutions, insertions or deletions occur within the regions outside the CDRs (i.e., FRs). A bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR, and a second and optionally a third antigen-binding domain that specifically binds to PD1, a first light chain variable domain (VL) sequence selected from the group consisting of SEQ ID NO:8 and SEQ ID NO:16, and a second light chain variable domain (VL) sequence selected from the group consisting of SEQ ID NO:24 and SEQ ID NO:32, including post-translational modifications of said sequences.

[0209] In another embodiment, there is provided a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second and optionally a third antigen-binding domain that specifically binds to PD1, the bispecific antibody comprising a VH sequence as in any of the embodiments provided above and a VL sequence as in any of the embodiments provided above. each including post-translational modifications of those sequences, A) a. SEQ ID NO: 7 and SEQ ID NO: 8, or b. SEQ ID NO:13 and SEQ ID NO:14 A first antigen-binding domain that specifically binds to TfR, comprising the VH sequence and the VL sequence of B) a. SEQ ID NO:23 and SEQ ID NO:24, or b. SEQ ID NO:31 and SEQ ID NO:32 and a second antigen-binding domain that specifically binds to PD1, and / or, if present, a third antigen-binding domain, comprising the VH and VL sequences of:

[0210] In a further embodiment of the invention, the bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second and optionally a third antigen-binding domain that specifically binds to PD1 according to any of the above embodiments is a monoclonal antibody, including a chimeric, humanized or human antibody. In one embodiment, the bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second and optionally a third antigen-binding domain that specifically binds to PD1 comprises at least one antibody fragment, such as an Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment.

[0211] In another embodiment, the antibody is a full-length antibody, such as an intact IgG1 antibody or other antibody class or isotype as defined herein. In certain embodiments, the antibody is of the IgG class. In another embodiment, the Fab fragment and / or Fc region of the antibody is of the IgG class. In certain embodiments, the antibody is of the IgG1 isotype. In another embodiment, the Fab fragment and / or Fc region of the antibody is of the IgG1 isotype.

[0212] In a further aspect, the antibody described herein is of the IgG1 isotype / subclass and comprises a constant heavy chain domain of SEQ ID NO:69 or SEQ ID NO:70, or a constant portion of the heavy chain amino acid sequence of SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39 or SEQ ID NO:41. In one aspect, furthermore, a C-terminal glycine (Gly446) is present. In one aspect, furthermore, a C-terminal glycine (Gly446) and a C-terminal lysine (Lys447) are present.

[0213] In another aspect, the invention relates to a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second antigen-binding domain that specifically binds to PD1, said bispecific antibody comprising a first heavy chain comprising an amino acid sequence having at least 95% sequence identity to sequence SEQ ID NO: 35, a first light chain comprising an amino acid sequence having at least 95% sequence identity to sequence SEQ ID NO: 36, a second heavy chain comprising an amino acid sequence having at least 95% sequence identity to sequence SEQ ID NO: 39, and a second light chain comprising an amino acid sequence having at least 95% sequence identity to sequence SEQ ID NO: 40.

[0214] In another aspect, the invention relates to a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second antigen-binding domain that specifically binds to PD1, said bispecific antibody comprising a first heavy chain comprising an amino acid sequence having at least 95% sequence identity to sequence SEQ ID NO: 37, a first light chain comprising an amino acid sequence having at least 95% sequence identity to sequence SEQ ID NO: 38, a second heavy chain comprising an amino acid sequence having at least 95% sequence identity to sequence SEQ ID NO: 39, and a second light chain comprising an amino acid sequence having at least 95% sequence identity to sequence SEQ ID NO: 40.

[0215] In another aspect, the invention relates to a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second antigen-binding domain that specifically binds to PD1, said bispecific antibody comprising a first heavy chain comprising an amino acid sequence having at least 95% sequence identity to sequence SEQ ID NO: 35, a first light chain comprising an amino acid sequence having at least 95% sequence identity to sequence SEQ ID NO: 36, a second heavy chain comprising an amino acid sequence having at least 95% sequence identity to sequence SEQ ID NO: 41, and a second light chain comprising an amino acid sequence having at least 95% sequence identity to sequence SEQ ID NO: 42.

[0216] In another aspect, the invention relates to a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second antigen-binding domain that specifically binds to PD1, said bispecific antibody comprising a first heavy chain comprising an amino acid sequence having at least 95% sequence identity to sequence SEQ ID NO: 37, a first light chain comprising an amino acid sequence having at least 95% sequence identity to sequence SEQ ID NO: 38, a second heavy chain comprising an amino acid sequence having at least 95% sequence identity to sequence SEQ ID NO: 41, and a second light chain comprising an amino acid sequence having at least 95% sequence identity to sequence SEQ ID NO: 42.

[0217] In another aspect, the invention provides a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second and a third antigen-binding domain that specifically bind to PD1, The present invention relates to a bispecific antibody comprising a first heavy chain (chain H) comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 59, a second heavy chain (chain K) comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 60, a first light chain (chain A) comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 57 and a second light chain (chain B) comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 58.

[0218] In a further aspect, the invention provides a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second and a third antigen-binding domain that specifically bind to PD1, The present invention relates to a bispecific antibody comprising a first heavy chain (chain H) comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 61, a second heavy chain (chain K) comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 60, a first light chain (chain A) comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 57, and a second light chain (chain B) comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 58.

[0219] In a further aspect, a bispecific antibody comprising a first antigen-binding domain that specifically binds TfR and a second and optionally a third antigen-binding domain that specifically binds PD1 according to any of the above aspects may incorporate any of the features, alone or in combination, as described in Sections 1-8 below.

[0220] 1. Antibody affinity In certain embodiments, the antibodies provided herein have a potency of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10-8 M or less, e.g., 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13 It has a dissociation constant (KD) of 1 M.

[0221] In one embodiment, K Dis measured using a BIACORE® surface plasmon resonance assay. For example, assays using a BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) are performed at 25° C. with antigen CM5 chips immobilized at about 10 response units (RU). In one embodiment, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen is diluted to 5 μg / ml (about 0.2 μM) with 10 mM sodium acetate (pH 4.8) and then injected at a flow rate of 5 μl / min to obtain about 10 response units (RU) of bound protein. After injection of antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS containing 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25° C. with a flow rate of approximately 25 μl / min. Association rates (k on ) and dissociation rate (k off ) is calculated. off / k on as the equilibrium dissociation constant (K D ) is calculated. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). If the association rate by the surface plasmon resonance assay described above is 10 6 M -1 s -1above, the association rate can be determined using a fluorescence quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm, emission = 340 nm, 16 nm band pass) of 20 nM anti-antigen antibody (Fab form) in PBS (pH 7.2) at 25°C in the presence of increasing concentrations of antigen, as measured in a spectrometer such as a spectrophotometer equipped with a stopped flow (Aviv Instruments) or an 8000 series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) equipped with a stirred cuvette.

[0222] Alternatively, K D is measured by radiolabeled antigen binding assay (RIA). In one embodiment, the RIA is performed using a Fab version of the antibody of interest and its antigen. For example, the solution binding affinity of a Fab for an antigen is measured by binding the Fab to a minimum concentration of ( 125 I) is measured by equilibrating with labeled antigen and then capturing bound antigen using a plate coated with anti-Fab antibody (see, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish assay conditions, MICROTITER® multiwell plates (Thermo Scientific) are coated overnight with 5 μg / mL of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6) and then blocked with 2% (w / v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23°C). In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM [ 125I]-antigen is mixed with serial dilutions of the Fab of interest (e.g., consistent with the evaluation of the anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight. However, incubation may continue for an extended period (e.g., about 65 hours) to ensure that equilibrium is reached. The mixture is then transferred to a capture plate and incubated at room temperature (e.g., 1 hour). The solution is then removed and the plate is washed 8 times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. Once the plate has dried, 150 μl / well of scintillant (MICROSCINT-20®; Packard) is added and the plate is counted for 10 minutes using a TOPCOUNT® gamma counter (Packard). The concentration of each Fab that results in 20% or less of maximum binding is selected for use in competitive binding assays.

[0223] 2. Antibody fragment In certain aspects, the antibodies provided herein are antibody fragments.

[0224] In one embodiment, the antibody fragment is a Fab, Fab', Fab'-SH, or F(ab')2 fragment, in particular a Fab fragment. Papain digestion of an intact antibody generates two identical antigen-binding fragments, called "Fab" fragments, containing the heavy and light chain variable domains (VH and VL, respectively), including the three CDRs in VH (CDR-H1, CDR-H2, CDR-H3), and the three CDRs in VL (CDR-L1, CDR-L2, CDR-L3), as well as the constant domain of the light chain (CL) and the first constant domain of the heavy chain (CH1). Thus, the term "Fab fragment" refers to an antibody fragment containing a light chain comprising a VL domain and a CL domain, and a heavy chain fragment comprising a VH domain and a CH1 domain. "Fab' fragments" differ from Fab fragments by the addition of residues at the carboxy terminus of the CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is a Fab' fragment in which the cysteine ​​residue in the constant domain bears a free thiol group. Pepsin treatment yields an F(ab')2 fragment that contains two antigen-binding domains (two Fab fragments) and a portion of the Fc region. See U.S. Patent No. 5,869,046 for a description of Fab and F(ab')2 fragments that contain salvage receptor binding epitope residues and have increased half-life in vivo. In one embodiment, the Fab fragment is of the IgG class.

[0225] In one embodiment, the antibody fragment is a diabody, triabody or tetrabody. A "diabody" is an antibody fragment that has two antigen-binding domains, which can be bivalent or bispecific. See, for example, EP 404,097; WO 1993 / 01161; Hudson et al., Nat.Med. 9:129-134 (2003); and Hollinger et al., Proc.Natl.Acad.Sci.USA 90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat.Med. 9:129-134 (2003).

[0226] In a further embodiment, the antibody fragment is a single chain Fab fragment. A "single chain Fab fragment" or "scFab" is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody heavy chain constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, said antibody domains and said linker having one of the following orders from N-terminus to C-terminus: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1 or d) VL-CH1-linker-VH-CL. In particular, said linker is a polypeptide of at least 30 amino acids, preferably 32 to 50 amino acids. The said single chain Fab fragment is stabilized by a native disulfide bond between the CL domain and the CH1 domain. In addition, these single-chain Fab fragments may be further stabilized by the insertion of cysteine ​​residues (e.g., at position 44 of the variable heavy chain and position 100 of the variable light chain, according to the Kabat numbering) to generate an interchain disulfide bond.

[0227] In another embodiment, the antibody fragment is a single chain variable fragment (scFv). A "single chain variable fragment" or "scFv" is a fusion protein of the variable domains of the heavy (VH) and light (VL) chains of an antibody connected by a peptidic linker. In particular, the linker is a short polypeptide of 10-25 amino acids, usually rich in glycine for flexibility and serine or threonine for solubility, which can connect either the N-terminus of the VH to the C-terminus of the VL, or vice versa. The protein retains the specificity of the original antibody despite the removal of the constant regions and the introduction of the linker. For a review of scFv fragments, see, for example, Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994). See also WO 93 / 16185 and U.S. Patent Nos. 5,571,894 and 5,587,458.

[0228] In another embodiment, the antibody fragment is a single domain antibody. A "single domain antibody" is an antibody fragment that contains all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 B1).

[0229] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and recombinant production by recombinant host cells (e.g., E. coli), as described herein.

[0230] 3. Chimeric and humanized antibodies In certain embodiments, the antibody provided herein is a chimeric antibody. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567, and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one example, the chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a non-human primate, such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In a further example, the chimeric antibody is a "class-switched" antibody whose class or subclass has been changed from that of the parent antibody. The chimeric antibody includes an antigen-binding fragment thereof.

[0231] In certain embodiments, the chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. In general, a humanized antibody comprises one or more variable domains in which the CDRs (or portions thereof) are derived from a non-human antibody and the FRs (or portions thereof) are derived from a human antibody sequence. Optionally, the humanized antibody also comprises at least a portion of a human constant region. In some embodiments, some FR residues of the humanized antibody are replaced by corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve the specificity or affinity of the antibody.

[0232] Humanized antibodies and methods for making them are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and are described, e.g., in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Pat. No. 5,821,337, U.S. Pat. No. 7,527,791, U.S. Pat. No. 6,982,321, and U.S. Pat. No. 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "resurfacing"); Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing a "guided selection" approach to FR shuffling).

[0233] Human framework regions that may be used for humanization include, but are not limited to, framework regions selected using the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from consensus sequences of human antibodies of particular subgroups of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening of FR libraries (see, e.g., Baca et al. al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996).

[0234] 4. Human antibodies In certain embodiments, the antibody provided herein is a human antibody. Human antibodies can be produced using various techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20: 450-459 (2008).

[0235] Human antibodies can be prepared by administering immunogens to transgenic animals that have been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigen exposure. Such animals typically contain all or part of the human immunoglobulin loci that replace endogenous immunoglobulin loci or are extrachromosomally or randomly integrated into the animal's chromosomes. In such transgenic mice, endogenous immunoglobulin loci are generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat.Biotech.23:1117-1125(2005). See also, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584, which describe XENOMOUSE™ technology; U.S. Patent No. 5,770,429, which describes HuMab® technology; U.S. Patent No. 7,041,870, which describes KM MOUSE® technology, and U.S. Patent Application Publication No. 2007 / 0061900, which describes VelociMouse® technology.) The human variable regions from intact antibodies generated by such animals can be further modified, for example, by combining with different human constant regions.

[0236] Human antibodies can also be produced by hybridoma-based methods. Human myeloma cell lines and mouse-human heteromyeloma cell lines for producing human monoclonal antibodies have been described. (See, for example, Kozbor J.Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp.51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J.Immunol., 147:86 (1991).) Human antibodies generated via human B-cell hybridoma technology are also described in Li et al., Proc.Natl.Acad.Sci.USA, 103:3557-3562 (2006). Additional methods include those described, for example, in U.S. Patent No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0237] Human antibodies can also be generated by isolating variable domain sequences selected from a phage display library of human origin. Such variable domain sequences may then be combined with the desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0238] 5. Library-derived antibodies In certain aspects, the antibodies provided herein are obtained from libraries. The antibodies of the present invention can be isolated by screening combinatorial libraries for antibodies with one or more desired activities. Methods for screening combinatorial libraries are, for example, reviewed in Lerner et al. in Nature Reviews 16:498-508 (2016). For example, various methods are known in the art for creating phage display libraries and screening such libraries for antibodies with desired binding properties. Such methods are reviewed, for example, in Frenzel et al. in mAbs 8:1177-1194 (2016); Bazan et al. in Human Vaccines and Immunotherapeutics 8:1817-1828 (2012) and Zhao et al. in Critical Reviews in Biotechnology 36:276-289 (2016), as well as Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001) and Marks and Bradbury in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003).

[0239] In a particular phage display method, repertoires of VH and VL genes can be cloned separately by polymerase chain reaction (PCR), randomly recombined in a phage library, which can then be screened for antigen-binding phage, as described by Winter et al. in Annual Review of Immunology 12:433-455 (1994). Phages typically display antibody fragments as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high affinity antibodies to immunogens without the need to construct hybridomas. Alternatively, naive repertoires can be cloned (e.g., from humans) to obtain a single source of antibodies against a wide range of non-self and even self antigens without immunization, as described by Griffiths et al. in EMBO Journal 12:725-734 (1993). In addition, naive libraries can also be synthetically generated by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences to encode highly variable CDR3 regions and achieve in vitro reordering, as described by Hoogenboom and Winter in Journal of Molecular Biology 227:381-388 (1992).Patent publications describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373; U.S. Patent No. 7,985,840; U.S. Patent No. 7,785,903 and U.S. Patent No. 8,679,490, as well as U.S. Patent Publication No. 2005 / 0079574, U.S. Patent Publication No. 2007 / 0117126, U.S. Patent Publication No. 2007 / 0237764 and U.S. Patent Publication No. 2007 / 0292936.

[0240] Further examples of methods known in the art for screening combinatorial libraries for antibodies with one or more desired activities include ribosome and mRNA display, and methods for antibody display and selection in bacteria, mammalian cells, insect cells or yeast cells.Methods for yeast surface display are, for example, outlined in Scholler et al.in Methods in Molecular Biology 503:135-56(2012) and Cherf et al.in Methods in Molecular biology 1319:155-175(2015) and Zhao et al.in Methods in Molecular Biology 889:73-84(2012).Methods for ribosome display are, for example, described in He et al.in Nucleic Acids Research 25:5132-5134(1997) and Hanes et al.in PNAS 94:4937-4942(1997).

[0241] Antibodies or antibody fragments isolated from a human antibody library are considered human antibodies or human antibody fragments herein.

[0242] 6. Multispecific antibodies In some embodiments, the bispecific antibodies provided herein are multispecific antibodies, e.g., trispecific or tetraspecific antibodies. A "multispecific antibody" is a monoclonal antibody that has binding specificities for at least two different sites, i.e., different epitopes on different antigens, or different epitopes on the same antigen. In certain embodiments, a multispecific antibody has three or more binding specificities. In certain embodiments, one of the binding specificities is for TfR, one of the binding specificities is for PD1, and the third specificity is for any other antigen. In certain embodiments, a bispecific antibody can bind to two (or more) different epitopes of TfR and / or PD1. Multispecific (e.g., bispecific) antibodies may be used to localize cytotoxic agents or cells to cells expressing PD1 and / or TfR. Multispecific antibodies may be prepared as full-length antibodies or antibody fragments.

[0243] Techniques for making multispecific antibodies include, but are not limited to, recombinant coexpression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983)), and "knobs-in-holes" engineering (see, e.g., U.S. Pat. No. 5,731,168, and Atwell et al., J. Mol. Biol. 270:26 (1997)). Multispecific antibodies can be produced by manipulating electrostatic steering effects to create antibody Fc-heterodimeric molecules (see, e.g., WO 2009 / 089004), cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science, 229:81 (1985)), using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992), and WO 2011 / 034605), using common light chain technology to circumvent light chain mispairing problems (see, e.g., WO 98 / 50431), using "diabody" technology to create bispecific antibody fragments (see, e.g., Hollinger et al., J. Immunol., 148(5):1547-1553 (1992) and WO 2011 / 034605), and using diabody technology to create bispecific antibody fragments (see, e.g., WO 98 / 50431). al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)), and using single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)), and by preparing trispecific antibodies as described, for example, in Tutt et al. J. Immunol. 147:60 (1991).

[0244] Also included herein are engineered antibodies having three or more antigen-binding domains, including, for example, "Octopus antibodies," or DVD-Igs (see, for example, WO 2001 / 77342 and WO 2008 / 024715). Other examples of multispecific antibodies having three or more antigen-binding domains can be found in WO 2010 / 115589, WO 2010 / 112193, WO 2010 / 136172, WO 2010 / 145792, and WO 2013 / 026831. Bispecific antibodies or antigen-binding fragments thereof also include "dual acting FAbs" or "DAFs" that contain a first antigen-binding domain that binds TfR and a second antigen-binding domain that binds PD1 and another different antigen, or two different epitopes on TfR and / or PD1 (see, e.g., U.S. Patent No. 2008 / 0069820 and WO 2015 / 095539).

[0245] Multispecific antibodies can also be provided in an asymmetric form with domain crossovers in one or more binding arms of the same antigen specificity, i.e., by exchanging VH / VL domains (see, e.g., WO 2009 / 080252 and WO 2015 / 150447), CH1 / CL domains (see, e.g., WO 2009 / 080253) or complete Fab arms (see, e.g., WO 2009 / 080251, WO 2016 / 016299, see also Schaefer et al, PNAS, 108 (2011) 1187-1191, and Klein at al., MAbs 8 (2016) 1010-20). In one embodiment, the multispecific antibody comprises a cross-Fab fragment. The term "cross-Fab fragment" or "xFab fragment" or "crossover Fab fragment" refers to a Fab fragment in which either the variable or constant regions of the heavy and light chains have been exchanged. A cross-Fab fragment comprises a polypeptide chain composed of a light chain variable region (VL) and a heavy chain constant region 1 (CH1) and a polypeptide chain composed of a heavy chain variable region (VH) and a light chain constant region (CL). Asymmetric Fab arms can also be engineered by introducing charged or uncharged amino acid mutations at the domain interface to direct correct Fab pairing. See, for example, WO 2016 / 172485.

[0246] A variety of additional molecular formats for multispecific antibodies are known in the art and are included herein (see, e.g., Spiess et al., Mol Immunol 67 (2015) 95-106).

[0247] Examples of bispecific antibody formats that may be useful for this purpose include, but are not limited to, so-called "BiTE" (bispecific T cell engager) molecules in which two scFv molecules are fused by a flexible linker (see, e.g., WO 2004 / 106381, WO 2005 / 061547, WO 2007 / 042261 and WO 2008 / 119567; Nagorsen and Bauerle, Exp Cell Res 317, 1255-1260 (2011)); diabodies (Holliger et al., Prot. Eng. 9, 299-305 (1996)) and derivatives thereof, such as tandem diabodies ("TandAbs"; Kipriyanov et al., J Mol Biol 1999, 2010 ... 293, 41-56 (1999); "DART" (dual affinity retargeting) molecules based on the diabody format but featuring a C-terminal disulfide bridge for further stabilization (Johnson et al., J Mol Biol 399, 436-449 (2010)), as well as the so-called triomabs, which are all-hybrid mouse / rat IgG molecules (reviewed in Seimetz et al., Cancer Treat. Rev. 36, 458-467 (2010)). Certain T cell bispecific antibody formats included herein are described in WO 2013 / 026833, WO 2013 / 026839, WO 2016 / 020309; Bacac et al., Oncoimmunology 5(8) (2016) e1203498.

[0248] In one embodiment a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second and optionally a third antigen-binding domain that specifically binds to PD1 comprises a) a first light chain and a first heavy chain of a full-length antibody that specifically binds to TfR; b) a second (modified) light chain and a second (modified) heavy chain of a full-length antibody that specifically binds to PD1, in which the variable domains VL and VH have been replaced with each other and / or the constant domains CL and CH1 have been replaced with each other; c) A triabody or a tetrabody in which one to four antigen-binding domains that specifically bind to one or two additional antigens (i.e., a third and / or fourth antigen) are fused to the C-terminus or N-terminus of the light chain or heavy chain of a) and / or b) via a peptidic linker.

[0249] In one embodiment a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second and optionally a third antigen-binding domain that specifically binds to PD1 comprises a) a first light chain and a first heavy chain of a full-length antibody that specifically binds to PD1; b) a second (modified) light chain and a second (modified) heavy chain of a full-length antibody that specifically binds to TfR, in which the variable domains VL and VH are replaced with each other and / or the constant domains CL and CH1 are replaced with each other, c) A triabody or a tetrabody in which one to four antigen-binding domains that specifically bind to one or two additional antigens (i.e., a third and / or fourth antigen) are fused to the C-terminus or N-terminus of the light chain or heavy chain of a) and / or b) via a peptidic linker.

[0250] The antibody of a) does not contain the modifications reported in b) and the heavy and light chains of a) are isolated chains.

[0251] In one embodiment the triabody or tetrabody comprises in c) one or two antigen binding domains which specifically bind to one or two further antigens.

[0252] In one embodiment, the antigen binding domain is selected from the group of a Fab fragment, an scFv fragment and an scFab fragment. In one embodiment, the antigen binding domain is a Fab fragment. In one embodiment, the antigen binding domain is an scFv fragment. In one embodiment, the antigen binding domain is an scFab fragment.

[0253] In one embodiment the antigen binding domain is fused to the C-terminus of the heavy chain of a) and / or b).

[0254] In one embodiment the triabody or tetrabody comprises in c) one or two antigen-binding domains which specifically bind to one further antigen.

[0255] In one aspect, the triabody or tetrabody comprises in c) two identical antigen-binding domains that specifically bind to a third antigen. In a preferred embodiment, such two identical antigen-binding domains are fused together to the C-terminus of the heavy chains of a) and b) via the same peptidic linker. In a preferred embodiment, the two identical antigen-binding domains are either Fab fragments, scFv fragments or scFab fragments.

[0256] In one aspect, the triabody or tetrabody comprises in c) two antigen-binding domains that specifically bind to a third and a fourth antigen. In one embodiment, the two antigen-binding domains are both fused to the C-terminus of the heavy chains of a) and b) via the same peptide connector. In a preferred embodiment, the two antigen-binding domains are either Fab fragments, scFv fragments or scFab fragments.

[0257] In one aspect, the bispecific antibody comprises: a) two light chains and two heavy chains of an antibody that specifically binds to a first antigen (comprising two Fab fragments); b) a bispecific tetravalent antibody comprising two additional Fab fragments of an antibody that specifically binds to a second antigen, the two additional Fab fragments being fused together via a peptidic linker to either the C-terminus or the N-terminus of the heavy chain of a); Here, the following modifications were made to the Fab fragment: (i) in both Fab fragments of a) or in both Fab fragments of b) the variable domains VL and VH are replaced by one another and / or the constant domains CL and CH1 are replaced by one another, or (ii) in both Fab fragments of a) the variable domains VL and VH are replaced by one another and the constant domains CL and CH1 are replaced by one another, and in both Fab fragments of b) the variable domains VL and VH are replaced by one another or the constant domains CL and CH1 are replaced by one another, or (iii) in both Fab fragments of a) the variable domains VL and VH are replaced by one another or the constant domains CL and CH1 are replaced by one another, and in both Fab fragments of b) the variable domains VL and VH are replaced by one another and the constant domains CL and CH1 are replaced by one another, or (iv) in both Fab fragments of a) the variable domains VL and VH are replaced by one another and in both Fab fragments of b) the constant domains CL and CH1 are replaced by one another; or (v) In both Fab fragments in a) the constant domains CL and CH1 are replaced by each other, and in both Fab fragments in b) the variable domains VL and VH are replaced by each other.

[0258] In one embodiment, said additional Fab fragments are both fused either to the C-terminus of the heavy chain of a) or to the N-terminus of the heavy chain of a) via a peptidic linker.

[0259] In one embodiment, said additional Fab fragments are both fused to either of the C-terminus of the heavy chain of a) via a peptidic linker.

[0260] In one embodiment said additional Fab fragments are both fused to the N-terminus of the heavy chain of a) via a peptide connector.

[0261] In one embodiment, the following modifications are made in the Fab fragments: in a) both Fab fragments or in b) both Fab fragments the variable domains VL and VH are replaced with each other and / or the constant domains CL and CH1 are replaced with each other.

[0262] In one aspect, the bispecific antibody comprises: a) a (modified) heavy chain of a first antibody that specifically binds to a first antigen and comprises a first VH-CH1 domain pair, wherein the N-terminus of a second VH-CH1 domain pair of the first antibody is fused to the C-terminus of the heavy chain via a peptidic linker; b) two light chains of the first antibody of a); and c) a (modified) heavy chain of a second antibody that specifically binds to a second antigen and comprises a first VH-CL domain pair, wherein the N-terminus of the second VH-CL domain pair of the second antibody is fused to the C-terminus of the heavy chain via a peptidic linker; d) a tetravalent antibody comprising two (modified) light chains of the second antibody of c), each of which comprises a CL-CH1 domain pair.

[0263] In one aspect, the bispecific antibody comprises: a) a heavy chain and a light chain of a first full-length antibody that specifically binds to a first antigen; b) the heavy and light chains of a second full-length antibody that specifically binds to a second antigen, wherein the N-terminus of the heavy chain is connected to the C-terminus of the light chain via a peptidic linker.

[0264] The antibody in a) does not contain the modifications reported in b) and the heavy and light chains are isolated chains.

[0265] In one aspect, the bispecific antibody comprises: a) a full-length antibody that specifically binds to a first antigen and that consists of two antibody heavy chains and two antibody light chains; b) an Fv fragment that specifically binds to a second antigen, the Fv fragment comprising a VH2 domain and a VL2 domain, both domains being connected to each other via disulfide bridges; Either the VH2 domain or the VL2 domain alone is fused via a peptidic linker to the heavy or light chain of a full-length antibody that specifically binds to a first antigen.

[0266] In a bispecific antibody, a) the heavy and light chains are separate chains.

[0267] In one embodiment, the other of the VH2 domain or the VL2 domain is not fused via a peptide linker to the heavy or light chain of the full length antibody that specifically binds to the first antigen.

[0268] In some embodiments, the first light chain comprises a VL domain and a CL domain, and the first heavy chain comprises a VH domain, a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain.

[0269] In one aspect, the bispecific antibody comprises: a) two Fab fragments that specifically bind to a first antigen; b) one CrossFab fragment that specifically binds to a second antigen, in which the CH1 and CL domains have been exchanged with each other; c) a trivalent antibody comprising one Fc region comprising a first Fc region heavy chain and a second Fc region heavy chain, This is a trivalent antibody in which the C-terminus of the CH1 domains of the two Fab fragments is connected to the N-terminus of a heavy chain Fc region polypeptide, and the N-terminus of the VH domain of the CrossFab fragment is connected to the C-terminus of the VH domain of one of the Fab fragments.

[0270] In one aspect, the bispecific antibody comprises: a) one Fab fragment that specifically binds to a first antigen; b) two CrossFab fragments that specifically bind to a second antigen, in which the CH1 and CL domains have been exchanged with each other; c) a trivalent antibody comprising one Fc region comprising a first Fc region heavy chain and a second Fc region heavy chain, This is a trivalent antibody in which the C-terminus of the CH1 domain of the Fab fragment is connected to the N-terminus of one of the heavy chain Fc region polypeptides, the C-terminus of the CL domain of one of the two CrossFab fragments is connected to the N-terminus of the other heavy chain Fc region polypeptide, and the C-terminus of the CH1 domain of the other of the two CrossFab fragments is connected to the N-terminus of the VH domain of the Fab fragment or the N-terminus of the VH domain of the CrossFab fragment.

[0271] In one aspect, the bispecific antibody comprises: a) a bivalent full-length antibody that specifically binds to a first antigen and that consists of two antibody heavy chains and two antibody light chains; b) a Fab fragment which specifically binds to a second antigen, comprising a VH2 domain and a VL2 domain comprising a heavy chain fragment and a light chain fragment, wherein in the light chain fragment the variable light chain domain VL2 is replaced by the variable heavy chain domain VH2 of said antibody, and in the heavy chain fragment the variable heavy chain domain VH2 is replaced by the variable light chain domain VL2 of said antibody, The heavy chain Fab fragment is inserted between the CH1 domain of one of the heavy chains of the full-length antibody and the respective Fc region of the full-length antibody, and the N-terminus of the light chain Fab fragment is conjugated to the C-terminus of the light chain of the full-length antibody that is paired with the heavy chain of the full-length antibody into which the heavy chain Fab fragment was inserted.

[0272] In one aspect, the bispecific antibody comprises: a) a bivalent full-length antibody that specifically binds to a first antigen and that consists of two antibody heavy chains and two antibody light chains; b) a Fab fragment which specifically binds to a second antigen, comprising a VH2 domain and a VL2 domain comprising a heavy chain fragment and a light chain fragment, wherein in the light chain fragment the variable light chain domain VL2 is replaced by the variable heavy chain domain VH2 of said antibody, and in the heavy chain fragment the variable heavy chain domain VH2 is replaced by the variable light chain domain VL2 of said antibody, and wherein the C-terminus of the heavy chain fragment of the Fab fragment is conjugated to the N-terminus of one of the heavy chains of a full-length antibody, and the C-terminus of the light chain fragment of the Fab fragment is conjugated to the N-terminus of the light chain of the full-length antibody which is paired with the heavy chain of the full-length antibody to which the heavy chain fragment of the Fab fragment is conjugated.

[0273] In one particular embodiment, a) one Fab fragment that specifically binds to the TfR; b) Two CrossFab fragments that specifically bind to PD1, in which the CH1 and CL domains have been exchanged with each other; c) a bispecific antibody is provided that is a trivalent antibody comprising one Fc region comprising a first Fc region heavy chain and a second Fc region heavy chain; The C-terminus of the CH1 domain of the Fab fragment is connected to the N-terminus of one of the heavy chain Fc region polypeptides, the C-terminus of the CH1 domain of the first CrossFab fragment is connected to the N-terminus of the other heavy chain Fc region polypeptide, and the C-terminus of the CH1 domain of the second CrossFab fragment is connected to the N-terminus of the VH domain of the Fab fragment or to the N-terminus of the VL domain of the CrossFab fragment. In a further embodiment, the Fc domain is an IgG Fc domain, in particular an IgG1 Fc domain or an IgG4 Fc domain. In a particular embodiment, the heavy chain of the bispecific antibody is of the gamma type (IgG), in particular of the gamma 1 type. In another particular embodiment, the light chain of the bispecific antibody is of the kappa (κ) and / or lambda (λ) subtype, based on the amino acid sequence of its constant domain.

[0274] In one particular embodiment, a) two CrossFab fragments that specifically bind to PD1, in which the VL and VH domains are exchanged with each other; b) one Fab fragment that specifically binds to TfR; c) a bispecific antibody is provided that is a trivalent antibody comprising one Fc region comprising a first Fc region heavy chain and a second Fc region heavy chain; The C-terminus of the CH1 domain of the first CrossFab fragment is connected to the N-terminus of one of the heavy chain Fc region polypeptides, the C-terminus of the CH1 domain of the Fab fragment is connected to the N-terminus of the other heavy chain Fc region polypeptide, and the C-terminus of the CH1 domain of the second CrossFab fragment is connected to the N-terminus of the VL domain of the CrossFab fragment or to the N-terminus of the VH domain of the Fab fragment. In a further embodiment, the Fc domain is an IgG Fc domain, in particular an IgG1 Fc domain or an IgG4 Fc domain. In a particular embodiment, the heavy chain of the bispecific antibody is of the gamma type (IgG), in particular of the gamma 1 type. In another particular embodiment, the light chain of the bispecific antibody is of the kappa (κ) and / or lambda (λ) subtype, based on the amino acid sequence of its constant domain.

[0275] In one aspect, a) a full-length antibody which specifically binds to PD1 and which consists of two antibody heavy chains and two antibody light chains, in which in the light chain the variable light domain VL is replaced by the variable heavy domain VH of said antibody, and in the heavy chain fragment the variable heavy domain VH is replaced by the variable light domain VL of said antibody, b) a Fab fragment that specifically binds to TfR; and The N-terminus of the heavy chain Fab fragment is conjugated to the C-terminus of one of the two heavy chains of the full-length antibody. In a further embodiment, the antibody and / or the Fab fragment is of the IgG class, in particular of the IgG1 or IgG4 isotype. In a particular embodiment, the heavy chain of the bispecific antibody is of the gamma type (IgG), in particular of the gamma 1 type. In another particular embodiment, the light chain of the bispecific antibody is of the kappa (κ) and / or lambda (λ) subtype, based on the amino acid sequence of its constant domain.

[0276] In one aspect, a) a full-length antibody that specifically binds to PD1 and is composed of two antibody heavy chains and two antibody light chains; b) a trivalent bispecific antibody comprising a CrossFab fragment that specifically binds to TfR, in which the CH1 domain and the CL domain are exchanged with each other; The N-terminus of the heavy chain CrossFab fragment is conjugated to the C-terminus of one of the two heavy chains of the full-length antibody.

[0277] 7. Antibody variants In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to change the binding affinity and / or other biological properties of the antibody. The amino acid sequence variants of the antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of residues in the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, so long as the final construct has the desired characteristics, e.g., antigen binding.

[0278] a) Substitution, insertion and deletion variants In certain embodiments, antibody variants with one or more amino acid substitutions are provided. Sites of interest for substitution mutagenesis include CDRs and FRs. Conservative substitutions are shown in Table 1 under the heading of "preferred substitutions". More substantial changes are provided in Table 2 under the heading of "exemplary substitutions" and are as further described below with reference to amino acid side chain classes. Amino acid substitutions may be introduced into the antibody of interest and the product screened for the desired activity, for example, retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC. TIFF2024528217000003.tif137170

[0279] Amino acids can be classified according to general side chain properties: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0280] Non-conservative substitutions involve exchanging a member of one of these classes for a member of another class.

[0281] Certain substitution variants involve substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variants selected for further testing will have modified (e.g., improved) specific biological properties (e.g., increased affinity, reduced immunogenicity) compared to the parent antibody and / or will have substantially retained specific biological properties of the parent antibody. An exemplary substitution variant is an affinity matured antibody, which can be conveniently generated using, for example, phage display-based affinity maturation techniques such as those described herein. In summary, one or more. CDR residues are mutated and the variant antibodies are displayed on phage and screened for specific biological activity (e.g., binding affinity).

[0282] For example, changes (e.g., substitutions) can be made to CDRs to improve antibody affinity. Such changes can be made to CDR "hot spots," i.e., residues encoded by codons that undergo frequent mutation during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or to residues that contact antigen, and the resulting variants VH or VL are tested for binding affinity. Affinity maturation by constructing and then reselecting from a secondary library is described, for example, in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001).). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then generated. This library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves a CDR-directed approach, in which several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.

[0283] In certain embodiments, substitutions, insertions or deletions may occur within one or more CDRs, so long as such changes do not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative changes (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made to the CDRs. Such changes may, for example, be outside of the antigen contact residues within the CDR. In the particular variant VH and variant VL sequences provided above, each CDR is unaltered or contains no more than one, two or three amino acid substitutions.

[0284] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis" as described in Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as arg, asp, his, lys and glu) is identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Further substitutions may be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively, or additionally, a crystal structure of an antigen-antibody complex may be used to identify contact points between the antibody and the antigen. Such contact and adjacent residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.

[0285] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertion variants of antibody molecules include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT (antibody-directed enzyme prodrug therapy)) or a polypeptide that increases the serum half-life of the antibody.

[0286] b) Glycosylation variants In certain embodiments, the antibodies provided herein are altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0287] If the antibody comprises an Fc region, the oligosaccharides attached thereto may be altered. Natural antibodies produced by mammalian cells typically comprise a branched biantennary oligosaccharide, generally linked to Asn297 of the CH2 domain of the Fc region by an N-linkage. See, for example, Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharides may comprise a variety of carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, as well as fucose attached to the GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modification of the oligosaccharides in the antibody of the present invention may be performed to create antibody variants with specific improved properties.

[0288] In one embodiment, antibody variants are provided that have nonfucosylated oligosaccharides, i.e., oligosaccharide structures that lack fucose attached (directly or indirectly) to the Fc region. Such nonfucosylated oligosaccharides (also referred to as "afucosylated" oligosaccharides) are in particular N-linked oligosaccharides that lack a fucose residue attached to the first GlcNAc of the stem of the biantennary oligosaccharide structure. In one embodiment, antibody variants are provided that have an increased proportion of nonfucosylated oligosaccharides in the Fc region compared to the native or parent antibody. For example, the proportion of nonfucosylated oligosaccharides can be at least about 20%, at least about 40%, at least about 60%, at least about 80%, or even about 100% (i.e., no fucosylated oligosaccharides are present). The percentage of nonfucosylated oligosaccharides is the (average) amount of oligosaccharides lacking a fucose residue compared to the sum of all oligosaccharides (e.g., complex, hybrid and high mannose structures) attached to Asn297, as measured by MALDI-TOF mass spectrometry, for example as described in WO 2006 / 082515. Asn297 refers to an asparagine residue located at about position 297 (Fc region residues in EU numbering) in the Fc region, although Asn297 may also be located ±3 amino acids upstream or downstream from position 297, i.e., between positions 294 and 300, due to minor sequence differences in antibodies. Such antibodies with an increased percentage of nonfucosylated oligosaccharides in the Fc region may have improved FcγRIIIa receptor binding and / or improved effector function, particularly improved ADCC function. See, for example, U.S. Patent No. 2003 / 0157108 and U.S. Patent No. 2004 / 0093621.

[0289] Examples of cell lines capable of producing antibodies with reduced fucosylation include Lec13 CHO cells, which are deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent No. 2003 / 0157108; and WO 2004 / 056312, especially Example 11), and knockout cell lines, such as alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614-622 (2004); Kanda, Y. et al. al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO 2003 / 085107), or cells with reduced or eliminated activity of GDP-fucose synthesis or transporter proteins (see, e.g., U.S. Patent No. 2004259150, U.S. Patent No. 2005031613, U.S. Patent No. 2004132140, U.S. Patent No. 2004110282).

[0290] In a further embodiment, the antibody variant is provided with bisected oligosaccharides, for example, biantennary oligosaccharides attached to the Fc region of the antibody are bisected by GlcNAc. Such antibody variants can have reduced fucosylation and / or improved ADCC function, as described above. Examples of such antibody variants are described, for example, in Umana et al., Nat Biotechnol 17, 176-180 (1999); Ferrara et al., Biotechn Bioeng 93, 851-861 (2006); WO 99 / 54342; WO 2004 / 065540, WO 2003 / 011878.

[0291] Also provided is an antibody variant that has at least one galactose residue in the oligosaccharide attached to Fc region.Such an antibody variant can have improved CDC function.Such an antibody variant is described, for example, in WO 1997 / 30087, WO 1998 / 58964 and WO 1999 / 22764.

[0292] c) Fc domain variants In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1 Fc region, a human IgG2 Fc region, a human IgG3 Fc region, or a human IgG4 Fc region) that contains an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0293] In certain embodiments, the present invention contemplates antibody variants that possess some, but not all, effector functions that make them desirable candidates for applications where in vivo antibody half-life is important, but where certain effector functions (such as complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC)) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / depleted CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to confirm that the antibody lacks FcγR binding (and thus likely lacks ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be used (see, e.g., ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (Cell Technology, Inc. Mountain View, CA; and CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.Alternatively, or in addition, the ADCC activity of the molecule of interest can be evaluated in vivo, for example, in an animal model as disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay can be performed to confirm that the antibody cannot bind to C1q and thus lacks CDC activity. See, for example, the C1q and C3c binding ELISA in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay may be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006); WO 2013 / 120929).

[0294] Antibodies with reduced effector function include those containing substitutions of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called "DANA" Fc variant with substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581).

[0295] Certain antibody variants have been described with improved or diminished binding to FcRs (see, e.g., U.S. Pat. No. 6,737,056, WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)).

[0296] In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, e.g., substitutions at positions 298, 333 and / or 334 (EU numbering of residues) of the Fc region.

[0297] In certain embodiments, the antibody variant comprises an Fc region having one or more amino acid substitutions that reduce FcγR binding, for example, at positions 234 and 235 (EU numbering of residues) of the Fc region. In one embodiment, the substitutions are L234A and L235A (LALA). In certain embodiments, the antibody variant further comprises D265A and / or P329G in the Fc region derived from a human IgG1 Fc region. In one embodiment, the substitutions are L234A, L235A and P329G (LALA-PG) in the Fc region derived from a human IgG1 Fc region. (See, e.g., WO 2012 / 130831). In another embodiment, the substitutions are L234A, L235A and D265A (LALA-DA) in the Fc region derived from a human IgG1 Fc region.

[0298] In some embodiments, modifications are made to the Fc region that result in altered (i.e., either improved or decreased) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0299] Antibodies with increased half-lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in U.S. Patent No. 2005 / 0014934 (Hinton et al.). These antibodies comprise an Fc region having one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include those having a substitution at one or more of Fc region residues: 238, 252, 254, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424 or 434, e.g., a substitution at Fc region residue 434 (see, e.g., U.S. Patent No. 7,371,826; Dall'Acqua, WF, et al. J. Biol. Chem. 281 (2006) 23514-23524).

[0300] The Fc region residues important for mouse Fc-mouse FcRn interaction have been identified by site-directed mutagenesis (see, e.g., Dall'Acqua, WF, et al. J. Immunol 169 (2002) 5171-5180). Residues I253, H310, H433, N434 and H435 (EU numbering of residues) are involved in the interaction (Medesan, C., et al., Eur. J. Immunol. 26 (1996) 2533; Firan, M., et al., Int. Immunol. 13 (2001) 993; Kim, JK, et al., Eur. J. Immunol. 24 (1994) 542). Residues I253, H310 and H435 were found to be important for the interaction of human Fc with mouse FcRn (Kim, JK, et al., Eur. J. Immunol. 29 (1999) 2819). Studies of the human Fc-human FcRn complex have shown that residues I253, S254, H435 and Y436 are important for the interaction (Firan, M., et al., Int. Immunol. 13 (2001) 993; Shields, RL, et al., J. Biol. Chem. 276 (2001) 6591-6604). Yeung, YA, et al. (J. Immunol. 182 (2009) 7667-7671) report and investigate various mutants of residues 248-259, 301-317, 376-382, and 424-437.

[0301] In certain embodiments, the antibody variant comprises an Fc region having one or more amino acid substitutions that reduce FcRn binding, such as substitutions at positions 253 and / or 310 and / or 435 (EU numbering of residues) of the Fc region. In certain embodiments, the antibody variant comprises an Fc region having amino acid substitutions at positions 253, 310 and 435. In one embodiment, the substitutions are I253A, H310A and H435A in the Fc region derived from human IgG1 Fc region. See, for example, Grevys, A., et al., J.Immunol.194(2015)5497-5508.

[0302] In certain embodiments, the antibody variant comprises an Fc region having one or more amino acid substitutions that reduce FcRn binding, such as substitutions at positions 310 and / or 433 and / or 436 (EU numbering of residues) of the Fc region. In certain embodiments, the antibody variant comprises an Fc region having amino acid substitutions at positions 310, 433 and 436. In one embodiment, the substitutions are H310A, H433A and Y436A in the Fc region derived from human IgG1 Fc region. (See, e.g., WO 2014 / 177460).

[0303] In certain embodiments, the antibody variant comprises an Fc region having one or more amino acid substitutions that increase FcRn binding, for example, substitutions at positions 252 and / or 254 and / or 256 (EU numbering of residues) of the Fc region. In certain embodiments, the antibody variant comprises an Fc region having amino acid substitutions at positions 252, 254 and 256. In one embodiment, the substitutions are M252Y, S254T and T256E in the Fc region derived from human IgG1 Fc region. For other examples of Fc region variants, see also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351.

[0304] The C-terminus of the heavy chain of the antibody as reported herein may be a complete C-terminus terminating in amino acid residue PGK. The C-terminus of the heavy chain may be a truncated C-terminus with one or two of the C-terminal amino acid residues removed. In a preferred embodiment, the C-terminus of the heavy chain is PG terminating in a truncated C-terminus. In one embodiment of all embodiments reported herein, an antibody comprising a heavy chain comprising a C-terminal CH3 domain as specified herein comprises a C-terminal glycine-lysine dipeptide (G446 and K447, EU index numbering of amino acid positions). In one embodiment of all embodiments reported herein, an antibody comprising a heavy chain comprising a C-terminal CH3 domain as specified herein comprises a C-terminal glycine residue (G446, EU index numbering of amino acid positions).

[0305] d) Cysteine ​​Engineered Antibody Variants In certain embodiments, it may be desirable to create cysteine ​​engineered antibodies, such as THIOMAB™ antibodies, in which one or more residues of an antibody are replaced by cysteine ​​residues. In certain embodiments, the replaced residues occur at accessible sites of the antibody. By replacing these residues with cysteine, reactive thiol groups are thereby placed at accessible sites of the antibody, which can be used to conjugate the antibody to other moieties, such as drug moieties or linker drug moieties, to create immunoconjugates, as further described herein. Cysteine ​​engineered antibodies can be generated, for example, as described in U.S. Pat. Nos. 7,521,541, 8,30,930, 7,855,275, 9,000,130, or WO2016040856.

[0306] e) Antibody derivative In certain aspects, the antibodies provided herein may be further modified to contain additional non-proteinaceous moieties that are known in the art and readily available. Moieties suitable for derivatization of antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol / propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in manufacturing due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and when multiple polymers are attached, they can be the same or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used therapeutically under defined conditions, etc.

[0307] 8. Immunoconjugates The invention also provides herein immunoconjugates comprising a bispecific antibody comprising a first antigen-binding domain that specifically binds TfR, and a second, and optionally a third antigen-binding domain that specifically binds PD1, conjugated (chemically linked) to one or more therapeutic agents, such as a cytotoxic agent, a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof) or a radioisotope.

[0308] In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC) in which the antibody is conjugated to one or more of the above therapeutic agents.The antibody is typically conjugated to one or more of the therapeutic agents using a linker.A review of ADC technology, including examples of therapeutic agents and drugs and linkers, is provided in Pharmacol Review 68:3-19 (2016).

[0309] In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including, but not limited to, diphtheria A chain, nonbinding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana proteins (PAPI, PAPII and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and the trichothecenes.

[0310] In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes are available for the generation of radioconjugates. Examples include At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212Radioactive conjugates, when used for detection, can include radioactive atoms for scintigraphic examination, such as tc99m or I123, or spin labels for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), again, such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

[0311] Conjugates of antibodies and cytotoxic agents can be made using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotides to antibodies. See WO 94 / 11026. The linker can be a "cleavable linker" that facilitates the release of the cytotoxic drug inside the cell. For example, acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers or disulfide-containing linkers (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Patent No. 5,208,020) can be used.

[0312] The immunoconjugates or ADCs herein expressly contemplate such conjugates prepared using cross-linking reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate), which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, Ill., USA).

[0313] B. Recombinant Methods and Compositions Antibodies can be produced using recombinant methods and compositions, for example, as described in U.S. Patent No. 4,816,567. For these methods, one or more isolated nucleic acids encoding the antibody are provided.

[0314] In the case of a natural antibody or a natural antibody fragment, two nucleic acids are required, one for the light chain or a fragment thereof and the other for the heavy chain or a fragment thereof. Such nucleic acids encode the amino acid sequence comprising the VL and / or the amino acid sequence comprising the VH of the antibody (e.g., the light and / or heavy chains of the antibody). These nucleic acids may be on the same expression vector or on different expression vectors.

[0315] In the case of a bispecific antibody having a heterodimeric heavy chain, four nucleic acids are required, one for the first light chain, one for the first heavy chain comprising the first heteromonomeric Fc region polypeptide, one for the second light chain, and one for the second heavy chain comprising the second heteromonomeric Fc region polypeptide. The four nucleic acids may be contained in one or more nucleic acid molecules or expression vectors. Such nucleic acids encode an amino acid sequence comprising a first VL, and / or an amino acid sequence comprising a first VH comprising the first heteromonomeric Fc region, and / or an amino acid sequence comprising a second VL, and / or an amino acid sequence comprising a second VH comprising the second heteromonomeric Fc region of the antibody (e.g., the first and / or second light chain and / or the first and / or second heavy chain of the antibody). These nucleic acids may be on the same expression vector or on different expression vectors, and usually these nucleic acids are located on two or three expression vectors, i.e. one vector may contain more than one of these nucleic acids. An example of these bispecific antibodies is CrossMab (see, for example, Schaefer, W. et al., PNAS, 108 (2011) 11187-1191). For example, according to the EU index numbering, one of the heteromonomer heavy chains contains a so-called "knob mutation" (T366W, and optionally one of S354C or Y349C), and the other contains a so-called "hole mutation" (T366S, L368A and Y407V, and optionally Y349C or S354C) (see, for example, Carter, P. et al., Immunotechnol. 2 (1996) 73).

[0316] In one aspect, an isolated nucleic acid encoding an antibody for use in the methods reported herein is provided.

[0317] In one aspect, there is provided a method of making a bispecific antibody comprising a first antigen binding domain that specifically binds TfR, and a second and optionally a third antigen binding domain that specifically binds PD1, the method comprising culturing a host cell comprising nucleic acid encoding the antibody as provided above under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell medium).

[0318] For recombinant production of a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second and optionally a third antigen-binding domain that specifically binds to PD1, for example, nucleic acids encoding the antibody as described above are isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of the antibody), or can be produced by recombinant methods, or can be obtained by chemical synthesis.

[0319] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies may be produced in bacteria, particularly if glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent No. 5,648,237, U.S. Patent No. 5,789,199 and U.S. Patent No. 5,840,523. (See also Charlton, KA, In: Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2003), pp. 245-254, which describes the expression of antibody fragments in E. coli.) After expression, the antibody may be isolated from the bacterial cell paste in a soluble fraction and may be further purified.

[0320] In addition to prokaryotes, useful eukaryotic microorganisms such as filamentous fungi or yeast, including fungal and yeast strains whose glycosylation pathways have been "humanized" resulting in the production of antibodies with partial or fully human glycosylation patterns, are also suitable cloning or expression hosts for antibody-encoding vectors. See Gerngross, TU, Nat. Biotech. 22 (2004) 1409-1414; and Li, H. et al., Nat. Biotech. 24 (2006) 210-215.

[0321] Suitable host cells for the expression of (glycosylated) antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for the transfection of Spodoptera frugiperda cells.

[0322] Plant cell cultures can also be used as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe the PLANTIBODIES™ technology for producing antibodies in transgenic plants).

[0323] Vertebrate cells may also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines include SV40 transformed monkey kidney CV1 line (COS-7); human embryonic kidney lines (e.g., 293 or 293T cells as described in Graham, FL et al., J. Gen Virol. 36 (1977) 59-74); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells as described in Mather, JP, Biol. Reprod. 23 (1980) 243-252); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells (e.g., Mather, JP et al., Annals NY Acad. Sci. 383 (1982) 44-68); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980) 4216-4220), and myeloma cell lines, such as Y0, NS0 and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

[0324] In one embodiment, the host cell is a eukaryotic cell, such as a Chinese Hamster Ovary (CHO) cell or a lymphoid cell (e.g., a Y0, NS0, Sp20 cell).

[0325] C. Assay Bispecific antibodies provided herein comprising a first antigen-binding domain that specifically binds to TfR and a second and optionally a third antigen-binding domain that specifically binds to PD1 can be identified, screened or characterized for their physical / chemical properties and / or biological activity by various assays known in the art.

[0326] 1. Binding and other assays In one embodiment, the antibodies of the invention are tested for their antigen binding activity by known methods such as, for example, ELISA, Western blot, and the like.

[0327] In another embodiment, a competitive assay may be used to identify antibodies that compete with mouse anti-human transferrin receptor antibody 128.1 (see WO 93 / 01819 and SEQ ID NOs: 64 and 65 for variable region sequences) for binding to TfR. In certain embodiments, such competing antibodies bind to the same epitope (e.g., a linear or conformational epitope) bound by mouse anti-human transferrin receptor antibody 128.1. Detailed exemplary methods for mapping antibody-binding epitopes are provided in Morris (1996) "Epitope Mapping Protocols", in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).

[0328] In an exemplary competitive assay, immobilized TfR is incubated in a solution containing a first labeled antibody that binds to TfR (e.g., mouse anti-human transferrin receptor antibody 128.1) and a second unlabeled antibody that is being tested for its ability to compete with the first antibody for binding to TfR. The second antibody may be present in a hybridoma supernatant. As a control, immobilized TfR is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow binding of the first antibody to TfR, excess unbound antibody is removed and the amount of label associated with immobilized TfR is measured. If the amount of label associated with immobilized TfR is substantially reduced in the test sample compared to the control sample, it indicates that the second antibody competes with the first antibody for binding to TfR. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0329] In another embodiment, a competitive assay may be used to identify antibodies that compete with, for example, nivolumab or pembrolizumab for binding to PD1. In certain embodiments, such competing antibodies bind to the same epitope (e.g., a linear or conformational epitope) that is bound by, for example, nivolumab or pembrolizumab. Detailed exemplary methods for mapping the epitope to which an antibody binds are provided in Morris (1996) "Epitope Mapping Protocols", in Methods in Molecular Biology vol.66 (Humana Press, Totowa, NJ).

[0330] In an exemplary competitive assay, immobilized PD1 is incubated in a solution containing a first labeled antibody (e.g., nivolumab or pembrolizumab) that binds to PD1 and a second unlabeled antibody that is being tested for its ability to compete with the first antibody for binding to PD1. The second antibody may be present in a hybridoma supernatant. As a control, immobilized PD1 is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow binding of the first antibody to PD1, excess unbound antibody is removed and the amount of label associated with immobilized PD1 is measured. If the amount of label associated with immobilized PD1 is substantially reduced in the test sample compared to the control sample, it indicates that the second antibody competes with the first antibody for binding to PD1. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0331] In another embodiment, a Jurkat cell assay is provided that allows the evaluation of the avidity-enhancing binding of bispecific anti-TfR anti-PD1 antibodies.To this end, NFAT-bla Jurkat cells that express PD1 at various levels are generated by transducing them with PD1 expression constructs by lentivirus.The Jurkat cells are contacted with bispecific antibodies and labeled.To evaluate whether binding depends on PD1 expression level, flow cytometry is used.This assay is described in more detail in Example 5.

[0332] 2. Activity Assay In one embodiment, an assay is provided for identifying a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second and optionally a third antigen-binding domain that specifically binds to PD1, which has biological activity. Biological activity may include, for example, the ability to enhance activation and / or proliferation of various immune cells, particularly T cells, secretion of immunomodulatory cytokines such as IFNγ or TNF-alpha, blocking the PD1 pathway, or killing tumor cells. Antibodies with such biological activity in vivo and / or in vitro are also provided.

[0333] In certain embodiments, the antibodies of the present invention are tested for such biological activity. In one embodiment, an immune cell assay is provided that measures activation of lymphocytes from one individual (donor X) to lymphocytes from another individual (donor Y). Mixed lymphocyte reaction (MLR) can show the effect of blocking PD1 pathway on lymphocyte effector cells. T cells in the assay are tested for activation measured by cytotoxic granzyme B release in the presence or absence of bispecific antibodies of the present invention. This assay is described in more detail in Example 13.

[0334] In another embodiment, a PD1 / PD-L1 blocking co-culture assay is provided that measures blocking of PD1 / PD-L1 mediated inhibition of TCR signaling between PD-L1 expressing CHO-K1 cells and PD1 expressing Jurkat-PD1-NFAT cells. Inhibition of TCR activation by PD1 signaling is measured by detecting expression of a reporter gene. This assay is described in further detail in Example 4.

[0335] In another embodiment, an internalization assay based on activated T cells is provided that allows for the determination of the internalization of bispecific anti-TfR anti-PD1 antibodies into cells. To this end, CD3 and CD28 activated CD4 T cells are first exposed to the antibodies at 4°C, then incubated at 37°C to allow internalization, followed by staining and fixing the cells. As a control, half of each sample is immediately washed, stained and fixed after exposure to the antibodies at 4°C (internalization at 4°C is negligible). Then, cells in both conditions (4°C and 37°C) are stained using a fluorescently labeled antibody that specifically binds to the bispecific anti-TfR anti-PD1 antibody. Fluorescence is detected using flow cytometry. Then, the geometric mean fluorescence intensity (GMFI) and the fluorescence labeled CD4 T cells are compared between the cells and the control cells. + Compare the frequency of T cells. Calculate the percentage of internalization using the following formula: 内部移行 %=100-((GMFI 蛍光性CD4+T 細胞 37℃ ÷GMFI 蛍光性CD4+T細胞 4℃ )*100)

[0336] This assay is described in further detail in Example 6.

[0337] D. Methods and Compositions for Diagnosis and Detection In certain embodiments, any of the bispecific antibodies provided herein, comprising a first antigen-binding domain that specifically binds to TfR and a second and optionally a third antigen-binding domain that specifically binds to PD1, are useful for detecting the presence of TfR or PD1 in a biological sample. As used herein, the term "detect" encompasses quantitative or qualitative detection. In certain embodiments, the biological sample comprises cells or tissues, such as immune cell infiltrates or T cell infiltrates, or tumor tissue.

[0338] In one embodiment, a bispecific antibody is provided comprising a first antigen-binding domain that specifically binds TfR and a second and optionally a third antigen-binding domain that specifically binds PD1 for use in a method of diagnosis or detection. In a further embodiment, a method is provided for detecting the presence of a bispecific antibody comprising a first antigen-binding domain that specifically binds TfR and a second and optionally a third antigen-binding domain that specifically binds PD1 in a biological sample. In certain embodiments, the method comprises contacting a biological sample with a bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second and optionally a third antigen-binding domain that specifically binds to PD1 antibody, as described herein, under conditions that permit binding of the bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second and optionally a third antigen-binding domain that specifically binds to PD1 antibody to TfR and / or PD1, and detecting whether a complex is formed between the bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second and optionally a third antigen-binding domain that specifically binds to TfR and / or PD1. Such methods may be in vitro or in vivo methods.

[0339] In certain embodiments, a labeled bispecific antibody is provided that comprises a first antigen-binding domain that specifically binds to TfR and a second and, optionally, a third antigen-binding domain that specifically binds to PD1. Labels include, but are not limited to, labels or moieties that are directly detected (such as fluorescent, chromophore, electron-dense, chemiluminescent and radioactive labels) and moieties, such as enzymes or ligands, that are indirectly detected, for example, by enzymatic reaction or molecular interaction. Exemplary labels include, but are not limited to, radioisotopes, 32 P, 14 C. 125 I, 3 H and 131I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases such as firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidases such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, dye precursors such as HRP, lactoperoxidase or microperoxidase, heterocyclic oxidases coupled with enzymes that use hydrogen peroxide to oxidize biotin / avidin, spin labels, bacteriophage labels, stable free radicals, and the like.

[0340] E. Pharmaceutical Compositions In a further aspect, a pharmaceutical composition is provided comprising any of the antibodies provided herein, e.g., for use in any of the following therapeutic methods. In one aspect, the pharmaceutical composition comprises any of the antibodies provided herein and a pharma- ceutically acceptable carrier. In another aspect, the pharmaceutical composition comprises any of the antibodies provided herein and at least one additional therapeutic agent, e.g., as described below.

[0341] Pharmaceutical compositions of bispecific antibodies comprising a first antigen-binding domain that specifically binds to TfR and a second and optionally a third antigen-binding domain that specifically binds to PD1, as described herein, are prepared by mixing such antibodies having the desired purity with one or more optional pharma- ceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) in the form of a lyophilized composition or an aqueous solution. Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed and include, but are not limited to, buffers, such as histidine, phosphates, citrates, acetates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); small molecule These include polypeptides of small amounts (less than about 10 residues); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharma- ceutically acceptable carriers herein further include interstitial drug dispersing agents, such as soluble neutral-active hyaluronidase glycoproteins (sHASEGPs), such as human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Halozyme, Inc.).Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one embodiment, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0342] Exemplary lyophilized antibody compositions are described in U.S. Pat...

Claims

1. A bispecific antibody comprising a first antigen-binding domain that specifically binds to TfR and a second antigen-binding domain that specifically binds to PD1.

2. 2. The bispecific antibody of claim 1, comprising a third antigen-binding domain that specifically binds to PD1.

3. 2. The bispecific antibody of claim 1 , wherein the first antigen-binding domain, the second antigen-binding domain, and / or, if present, the third antigen-binding domain are Fab fragments.

4. 4. The bispecific antibody of claim 3, comprising an Fc domain composed of a first and a second subunit, wherein one or more of the Fab fragments are fused to the Fc domain, and wherein the Fc domain is an IgG Fc domain, in particular an IgG1 Fc domain or an IgG4 Fc domain.

5. 2. The antibody of claim 1, wherein the first antigen-binding domain, the second antigen-binding domain, and, if present, the third antigen-binding domain are each Fab fragments, and the antibody comprises an Fc domain composed of first and second subunits, and wherein (i) the second antigen-binding domain is fused at the C-terminus of its Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding domain and the first antigen-binding domain is fused at the C-terminus of its Fab heavy chain to the N-terminus of the first subunit of the Fc domain, or (ii) the first antigen-binding domain is fused at the C-terminus of its Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding domain and the second antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and the third antigen-binding domain, if present, is fused at the C-terminus of its Fab heavy chain to the N-terminus of the second subunit of the Fc domain.

6. the first, second, and third antigen-binding domains are each Fab fragments, and the antibody comprises an Fc domain composed of a first and a second subunit; a first antigen-binding domain fused at the C-terminus of its Fab heavy chain to the N-terminus of the first subunit of the Fc domain; a second antigen-binding domain fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain; The antibody of claim 2, wherein the third antigen-binding domain is fused at the N-terminus of its Fab heavy chain to the C-terminus of the first or second subunit of the Fc domain.

7. a) binding to TfR and PD1 on the surface of a cell expressing TfR and PD1, and the bispecific antibody being internalized into said cell; and / or b) binding of the bispecific antibody to TfR and PD1 displayed on the surface of said cells results in depletion of PD1 from the surface of said cells; The bispecific antibody of claim 1.

8. comprising at least two heavy chains and at least two light chains; a) the heavy chains of the bispecific antibody are of the gamma type (IgG), in particular of the gamma 1 type, and / or b) The bispecific antibody of claim 1, wherein the light chains of the bispecific antibody are selected from the kappa (κ) and / or lambda (λ) subtypes.

9. The Fc domain is i. one or more amino acid substitutions that reduce binding to Fc receptors, particularly Fcγ receptors, and / or ii. The bispecific antibody of claim 4, comprising a modification that promotes association of the first and second subunits of the Fc domain.

10. 4. The bispecific antibody of claim 3, wherein in one of the Fab fragments the variable domains VL and VH are replaced by each other so that the VH domain is part of a light chain and the VL domain is part of a heavy chain.

11. a first antigen-binding domain that specifically binds to TfR, i. a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3 a heavy chain variable domain (VH) comprising: d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6 a light chain variable domain (VL) comprising or ii. a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9; b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11 a heavy chain variable domain (VH) comprising: d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 12; e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 14 2. The bispecific antibody of claim 1, comprising a light chain variable domain (VL) comprising:

12. the second antigen-binding domain that specifically binds to PD1 and / or, if present, the third antigen-binding domain, i. a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 17; b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 18, and c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 19 a heavy chain variable domain (VH) comprising: d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 20; e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 21, and f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 22 a light chain variable domain (VL) comprising or ii. a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 25; b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 26, and c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 27 a heavy chain variable domain (VH) comprising: d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 28; e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 29, and f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 30 2. The bispecific antibody of claim 1, comprising a light chain variable domain (VL) comprising:

13. i. the first antigen-binding domain that specifically binds to TfR comprises: a) a VH domain comprising the amino acid sequence of SEQ ID NO: 7 and a VL domain comprising the amino acid sequence of SEQ ID NO: 8, or b) a VH domain comprising the amino acid sequence of SEQ ID NO: 15 and a VL domain comprising the amino acid sequence of SEQ ID NO: 16; and ii. the second antigen-binding domain and / or, if present, the third antigen-binding domain that specifically binds to PD1 a) a VH domain comprising the amino acid sequence of SEQ ID NO: 23 and a VL domain comprising the amino acid sequence of SEQ ID NO: 24, or b) A bispecific antibody according to claim 1, comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 31 and a VL domain comprising the amino acid sequence of SEQ ID NO:

32.

14. 14. A pharmaceutical composition comprising the bispecific antibody of any one of claims 1 to 13 and a pharmaceutically acceptable carrier.

15. For use in the prevention or treatment of cancer, (i) a bispecific antibody according to any one of claims 1 to 13, or (ii) A pharmaceutical composition comprising the bispecific antibody of any one of claims 1 to 13 and a pharmaceutically acceptable carrier.

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