Use of FAP-CD40 bispecific antibody for the modulation of the tumor microenvironment

Abstract

The invention relates to bispecific FAP-CD40 antibodies and its use for increasing the density of intratumoral DC-LAMP+ DCs and B cells and activating the immune system in the tumor so that the tumor will be more sensitive to treatment with anti-cancer agents.

Description

P39631 Use of FAP-CD40 bispecific antibody for the modulation of the tumor microenvironment Field of the Invention The invention relates to bispecific FAP-CD40 antibodies and its use for increasing the density of intratumoral DC-LAMP+ DCs and B cells and activating the immune system in the tumor so that the tumor is more sensitive to treatment with anti-cancer agents. The invention also relates to a new gene signature indicating FAP-CD40 mediated immune activation. Background of the Invention Cancer immunotherapy modulates the immune system and stimulates immune defenses against tumors. Despite the advances in cancer immunotherapies with improved clinical outcome, only a minority of patients benefit from these therapies and the responses are mostly of limited duration. Inefficient antigen presentation and T-cell priming in the intratumoral microenvironment are hypothesized to limit the efficacy of current immunotherapies such as checkpoint inhibitors (CPIs) or cancer vaccines. Although the addition of CPIs to chemotherapy has provided new options in the treatment of cancer, they did not show clinical benefit in immune desert tumors or in those tumors with dysfunctional or exhausted T cells. Dendritic cells (DCs) promote and coordinate the immune system and thereby play a crucial role in the ability of the immune system to recognize and eliminate tumor cells. As part of the innate system, DCs produce protective cytokines such as IL-12 and IL-6 and growth factors in response to “danger” signals that modulate ongoing immune responses, but they are also antigen-presenting cells (APCs) and internalize tumor antigens that are released into the tumor microenvironment (TME). After DC maturation, they efficiently present these peptides through the major histocompatibility complex (MHC) molecules MHC-I and MHC-II to naïve T- cells in the lymphoid tissues, inducing adaptive responses mediated by CD4+ and CD8+ T-cells. By interacting with DCs, naïve T-cells differentiate into effector T-cells with several functions, which ultimately results in the generation of a tumor-specific cellular and humoral response. In addition, DCs interact with other cells of the innate immune system, such as natural killers (NKs), macrophages or mast cells, resulting in the generation of a powerful and complete immune response (Hato et al., Cancers 2024, 16(5), 981). CD40 is a co-stimulatory receptor of the tumor necrosis factor (TNF) alpha receptor family which is expressed on antigen-presenting cells (APCs), such as dendritic cells (DCs), macrophages, and B cells, and plays a central role in the generation of an immune response (Elgueta et al., Immunol. Rev.2009, 229(1), 152-172). Activation of CD40 with therapeutic DK / 05.08.2025agonistic monoclonal antibodies (mAbs) has thus been identified as a promising approach to enhance anti-cancer immunity, prompting the investigation of several CD40 agonistic antibodies. However, in the past two decades, the clinical development of conventional CD40 agonists encountered several challenges, including systemic toxicities due to on-target off tumor CD40 activation and insufficient agonist activity, all of which partly contributed to the limited clinical efficacy observed so far. Bispecific antigen binding molecules capable of specific binding to CD40 and Fibroblast Activation Protein (FAP) are described in WO 2018 / 185045 A1, WO 2020 / 070041 A1 or WO 2020 / 070035 A1. These molecules combine a moiety capable of binding to FAP with a moiety capable of agonistic binding to CD40, wherein the activation of APCs through CD40 is provided by cross-linking through FAP expressed on tumor stroma cells and potentially also through FAP intermediately expressed in secondary lymphoid tissues. In contrast to bispecific antigen binding molecules capable of specific binding to CD40 and to immune checkpoint receptors on activated T cells, such as CTLA-4 or PD-1, targeting to a tumor target such as FAP enables CD40- mediated APC activation mainly in the tumor stroma and tumor-draining lymph nodes where fibroblasts express increased levels of FAP compared to other tissues. The fibroblast activation protein alpha (FAP)-targeted CD40 (FAP-CD40) agonist antibodies such as RO7300490 (FAP- CD40) may thus be able to trigger the CD40 receptor not only effectively, but also very selectively via crosslinking to FAP-expressing cells (Sum et al, Clin Cancer Res 2021, 27(14), 4036-4053). This is a key improvement over earlier generation CD40 agonists, considering the wide expression of CD40 in the periphery, e.g. on circulating B cells, platelets and endothelial cells. The membrane bound FAP, which is specifically expressed by cancer-associated fibroblasts in the tumor stroma of most human epithelial malignancies acts as a tumor targeting site for FAP-CD40, thereby allowing CD40 receptor clustering on the surface of tumor infiltrating APCs, resulting in downstream signaling to promote APC activation. Key target cells for FAP-CD40-mediated activation are conventional type 1 DCs (cDC1) and dendritic cell-lysosomal associated membrane protein-positive (DC-LAMP+) DCs. The cDC1 subset is known for its high capacity in antigen-cross presentation (den Haan et al., J. Exp. Med.2000, 192(12), 1685-1696, Böttcher et al., Trends in Cancer 2018, 4(11), 784-792), a process involving uptake of extracellular antigen and its subsequent presentation to CD8+ cytotoxic T lymphocytes (CTL). DC-LAMP is a marker for maturation of DCs and it is essential to promote effective antigen presentation. DCs of this subtype have been described as the most potent in terms of their T cell activation potential (de Saint-Vis et al., Immunity 1998, 9(1), 325- 336; Arruda et al., J. Immunology 2006, 177(4), 2265-2275). The presence of both these DCs subsets in tumor tissue has been associated with a better prognosis and better outcomes in cancer immunotherapy, indicative of their role in presenting tumor antigens to T cells and promotingimmune cell mediated antitumor responses (Ladányi et al., Cancer Immunol. Immunother.2007, 56, 1459–1469, Böttcher et al., Trends in Cancer 2018, 4(11), 784-792). There is a high need for agents that increase the density and maturation of these DC subsets and thereby enhance the generation of effective anti-tumor immune responses in combination with other anticancer reagents, promoting either antigen release or enhancing T cell activation. Summary of the Invention By preferentially targeting tumor infiltrating DCs, it has been found that FAP-CD40 activates and enhances the maturation of DC subsets, which can ultimately lead to an increased tumor antigen presentation to CD8+ cytotoxic T lymphocytes (CTLs). Treatment with FAP- CD40 resulted in a remarkable increase of (DC-LAMP+) DC density and B cell abundance and their organization into tertiary lymphoid structures (TLS). Thus, provided herein is a bispecific agonistic CD40 antibody comprising at least one antigen binding domain capable of specific binding to Fibroblast Activation Protein (FAP) (FAP-CD40 antibody) for use in treating cancer in combination with at least one anti-cancer agent, wherein the bispecific FAP-CD40 antibody increases the density of intratumoral DC- LAMP+ DCs and activates the immune system in the tumor microenvironment so that it will be more sensitive to treatment with the anti-cancer agent, meaning that the treatment with the anti- cancer agent can be more effective. In one aspect of the invention, the bispecific FAP-CD40 antibody comprises at least one antigen binding domain capable of specific binding to CD40 comprising a heavy chain variable region VH (CD40) comprising the amino acid sequence of SEQ ID NO:1 and a light chain variable region VL (CD40) comprising the amino acid sequence of SEQ ID NO:2, and at least one antigen binding domain capable of specific binding to Fibroblast Activation Protein (FAP) comprising (a) a VH (FAP) comprising the amino acid sequence of SEQ ID NO:3 and a VL (FAP) comprising the amino acid sequence of SEQ ID NO:4, (b) a VH (FAP) comprising the amino acid sequence of SEQ ID NO:5 and a VL (FAP) comprising the amino acid sequence of SEQ ID NO:6, or (c) a VH (FAP) comprising the amino acid sequence of SEQ ID NO:7 and a light chain variable region VL (FAP) comprising the amino acid sequence of SEQ ID NO:8. In one aspect of the invention, the bispecific FAP-CD40 antibody comprises a Fc domain of human IgG1 subclass with the amino acid mutations L234A, L235A and P329G (numbering according to Kabat EU index). Alternatively, it may comprise a Fc domain of human IgG1 or IgG4 subclass with one or more amino acid substitutions that reduce the binding affinity of the antibody to an Fc receptor and / or effector function.In one particular aspect, the bispecific FAP-CD40 antibody comprises one antigen binding domain capable of specific binding to FAP comprising the VH (FAP) comprising the amino acid sequence of SEQ ID NO:3 and the VL (FAP) comprising an amino acid sequence of SEQ ID NO:4. In one aspect, the bispecific FAP-CD40 antibody comprises (a) at least two Fab fragments capable of specific binding to CD40 fused at its C-terminus to the N-terminus of a Fc region, and (b) a cross-fab fragment capable of specific binding to FAP fused to the C-terminus of the Fc region. More particularly, the bispecific FAP-CD40 antibody comprises a cross-fab fragment capable of specific binding to FAP, wherein the VH-Ckappa chain of the cross-fab fragment is fused to the C-terminus of the Fc region. In one particular aspect of the invention, the bispecific FAP-CD40 antibody comprises two light chains, each comprising the amino acid sequence of SEQ ID NO:9, one light chain comprising the amino acid sequence of SEQ ID NO:10, a first heavy chain comprising the amino acid sequence of SEQ ID NO:11, and a second heavy chain comprising the amino acid sequence of SEQ ID NO:12. In another aspect of the invention, at least one anti-cancer agent is an agent blocking PD- L1 / PD-1 interaction. In one aspect, the agent blocking PD-L1 / PD-1 interaction is an anti-PD-L1 antibody or an anti-PD1 antibody. More particularly, the agent blocking PD-L1 / PD-1 interaction is an anti-PD-L1 antibody, in particular an anti-PD-L1 antibody selected from the group consisting of atezolizumab, durvalumab, pembrolizumab and nivolumab. In one specific aspect, the agent blocking PD-L1 / PD-1 interaction is atezolizumab (MPDL3280A, RG7446). In another specific aspect, the agent blocking PD-L1 / PD-1 interaction is pembrolizumab. In another aspect of the invention, at least one anti-cancer agent is a cancer vaccine. In a further aspect, the increase of the density of intratumoral DC-LAMP+ DCs and B cells is determined in a tumor tissue sample by immunofluoresence (IF) analysis. In particular, the tumor tissue sample is from tumor biopsy of a patient being treated with the bispecific FAP- CD40 antibody. In another aspect, the activation of the immune system in the tumor is determined by detecting the expression level of one or more genes selected from a group consisting of NFKBID, NFKBIE, CD40, FAS, SEMA7a, CCL22, TRAF1, BCL2A1, IL1A, HSPA1A, CD83, IL4I1, CD80, CD86, CXCL10, and CFB from a tumor tissue sample and comparing the expression level of these genes to a reference level, whereby an increase in the expression level is indicative for the activation of the immune system in the tumor. In particular, detecting the expression level is performed by RNA sequencing analysis. Preferably, the activation of the immune system in the tumor is determined by detecting the expression level of all genes selected from the group consisting of FAS, SEMA7A, CCL22, TRAF1, BCL2A1, IL4I1 and CFB.In one additional aspect, the invention relates to an in vitro method of monitoring if apatient having a tumor is responsive to cancer immunotherapy, the method comprising determining the activation of the immune system in a tumor tissue sample obtained from the patient having received a bispecific FAP-CD40 antibody in a first treatment regimen, wherein the activation of the immune system in the tumor is characterized by detecting the expression level of all genes selected from the group consisting of FAS, SEMA7A, CCL22, TRAF1, BCL2A1, IL4I1 and CFB and wherein the method further comprises a step of comparing the expression level of the genes to a reference level, whereby an increased expression level indicates that the tumor will be more sensitive to treatment with an anti-cancer agent in a second treatment regimen. The first treatment regimen comprises one or more cycles of treatment with the bispecific FAP-CD40 antibody alone or in combination with at least one other anti-cancer agent. The second treatment regimen comprises treatment with the anti-cancer agent. These and other aspects are further described in the detailed description below. Brief Description of the Figures Figures 1A to 1C show that FAP-CD40 increased the density and activation of mature DC-LAMP+ DCs in tumors. Shown is the infiltration status of DC-LAMP+ DCs (Fig.1A) and Clec9a+ DCs (Fig.1B) in baseline (BL) and on treatment (OT) paired tissue biopsies at both 140 mg and 550 mg doses. Exemplary multiplex-Immunofluorescence images from two patients (pt. 1008 -Breast cancer, skin lesion- and pt.1000 -Mesothelioma, lung lesion) showing DC-LAMPdensity in paired BL and OT biopsy tissues are displayed in Fig. 1C. Immunofluorescenceimages from pt.1000 also capture increases of DC-LAMP as well as the activation marker CD86 on treatment (Fig.1C, right).Green circles: DC-LAMP+ single positive cells. White circles: DC- LAMP+ CD86+ double positive cells. Purple: DC-LAMP, Yellow: CD86, Blue: DAPI (nuclei). Pt.: patient. FC: fold-change on treatment versus baseline. Figures 2A to 2C show that FAP-CD40 increased B cell density and organization in TLS-like foci in tumors. Fig. 2A shows the infiltration status of B cells in baseline (BL) and ontreatment (OT) paired tissue biopsies. Fig. 2B displays Immunofluorescence images from twopatients (pt): pt.1008 (breast cancer, skin lesion) and pt.1006 (mesothelioma, abdomen lesion) capturing distribution and frequency of B cell foci in paired baseline and on treatment biopsy tissues. Fig.2C shows DC-LAMP+ DCs and B cells in pt.1006 (mesothelioma, abdomen lesion). Total view on biopsy sample and enlarged view on individual foci, displaying the increased DC-LAMP+ DC and B cell abundance as well as the colocalization of both cell types after treatment. Tumour area annotated in red. Blue circles: B cells distributed in foci. Green: CD20; Purple: DC-LAMP; Blue: DAPI (nuclei). BL: baseline; OT: on-treatment. Pt.: patient. FC: fold-change on treatment versus baseline.Figure 3 shows the study design of an in vivo mouse study to evaluate the gene expressionupon treatment with an anti-FAP / anti-CD40 bispecific mouse surrogate molecule (P1AE2302- 039) in MC38-FAP tumor samples by NanoString. The timeline describes the treatment and sacrifice timepoint and the table describes the details of the treatment groups and the dosing. The white arrow depicts administration of P1AE2302-039. Figure 4 shows the study design to evaluate the differential gene expression on sorted cell populations isolated from KPC-4662-huCEA tumor bearing mice upon treatment with an anti- FAP / anti-CD40 bispecific mouse surrogate molecule (P1AE5394-122) by single cell RNA sequencing (scRNA-seq) (subsequently referred to as exp1). The timeline describes the treatment and sacrifice timepoints and the table describes the details of the treatment groups and the dosing. The white arrow depicts administration of P1AE5394-122. Figure 5 shows a schematic representation of the Perfusion-based bioreactor system andexperimental procedure to evaluate the differential gene expression on sorted cell populationsisolated from ex vivo human tumor explants upon treatment with an anti-FAP / anti-CD40bispecific human surrogate molecule (P1AE2423-020) by scRNA-seq (subsequently referred to as exp2 / exp3). The table describes the details of the human tumor tissues, culture conditions and sorted cell populations used for analysis. Figure 6 shows RNA expression levels (average expression in colors and fraction of positives as dot sizes) for genes induced upon treatment with a mouse surrogate of FAP-CD40 (P1AE5394-122) across cell types as measured in mouse tumor samples (exp1, tumor fraction, all identified cell types included), as measured by scRNA-seq. Figures 7A and 7B show changes in RNA expression levels (log2 fold-change, FC) insamples treated with a mouse surrogate of FAP-CD40 versus control samples (T_vs_C) at two distinct timepoints (24h and 48h) as measured in mouse tumor samples across all cells determined transcriptionally to be (A) immune cells (exp1, tumor fraction, immune cells) (Fig.7A), and (B) dendritic cells (Fig.7B) (exp1, tumor fraction, dendritic cells), as measured by scRNA-seq. FCs of 1.5 (black) and 2 (grey) are indicated with dotted lines. Figures 8A and 8B show changes in RNA expression levels induced by treatment with a human surrogate of FAP-CD40 (P1AE2423-020) in human tumor explants for selected genes, asmeasured by scRNA-seq. Fig. 8A shows changes in gene expression (log2FC) in treatmentversus control at three distinct timepoints (24h / T24, 48h / T48, 72h / T72) in transcriptionally identified B cells from a single donor (Pat 5; exp2, tumor explants, B cells); Fig.8B shows changes in gene expression (log2FC) in treatment versus control in transcriptionally identified myeloids cells of human tumor explants derived from four distinct donors (Pat 1-4) (exp3, tumor explants, myeloid cells); FCs of 1.5 (black) and 2 (grey) are indicated with dotted lines.Figures 9A to 9C shows differences in RNA expression levels between on treatment and baseline tumor biopsies from RO7300490 (FAP-CD40) treated patients as measured by bulk RNA sequencing. Mean gene expression difference (mean_log2FC) for 3 distinct signatures (B cells, FAP-CD40 activity [FAP_CD40_up_i], DC-LAMP+ dendritic cells [cDC_CCR7]) aredisplayed across all patients (Fig. 9A), or two distinct dose groups (Fig. 9B and Fig. 9C,respectively). Detailed Description Definitions Unless defined otherwise, technical and scientific terms used herein have the same meaning as generally used in the art to which this invention belongs. For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. As used herein, the terms "antigen binding molecule" or “antibody” are used interchangably and refer in their broadest sense to a molecule that specifically binds an antigenic determinant. Examples of antigen binding molecules are antibodies, bi-or multispecific antibodies, antibody fragments and scaffold antigen binding proteins. The term "antibody" herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity. As used herein, the term “antigen binding domain capable of specific binding to a target cell antigen” or "moiety capable of specific binding to a target cell antigen" refers to a polypeptide molecule that specifically binds to an antigenic determinant. In one aspect, the antigen binding domain is able to activate signaling through its target cell antigen. In a particular aspect, the antigen binding domain is able to direct the entity to which it is attached (e.g. the CD40 agonistic antibody) to a target site, for example to a specific type of tumor cell or tumor stroma bearing the antigenic determinant. Antigen binding domains capable of specific binding to a target cell antigen include antibodies and fragments thereof as further defined herein. In addition, antigen binding domains capable of specific binding to a target cell antigen include scaffold antigen binding proteins as further defined herein, e.g. binding domains which are based on designed repeat proteins or designed repeat domains (see e.g. WO 2002 / 020565). In particular, the antigen binding domain capable of specific binding to a target cell antigen is an antigen binding domain capable of specific binding to Fibroblast Activation Protein (FAP). In relation to an antibody or fragment thereof, the term "antigen binding domain capable of specific binding to a target cell antigen" refers to the part of the molecule that comprises the area which specifically binds to and is complementary to part or all of an antigen. An antigen bindingdomain capable of specific antigen binding may be provided, for example, by one or more antibody variable domains (also called antibody variable regions). Particularly, an antigen binding domain capable of specific antigen binding comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH) of an antibody. In another aspect, the "antigen binding domain capable of specific binding to a target cell antigen" can also be a Fab fragment or a cross-Fab fragment. The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind the same epitope, except for possible variant antibodies, e.g. containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. The term “monospecific” antibody as used herein denotes an antibody that has one or more binding sites each of which bind to the same epitope of the same antigen. The term “bispecific” means that the antigen binding molecule is able to specifically bind to at least two distinct antigenic determinants. Typically, a bispecific antigen binding molecule comprises two antigen binding sites, each of which is specific for a different antigenic determinant. In certain embodiments the bispecific antigen binding molecule is capable of simultaneously binding two antigenic determinants, particularly two antigenic determinants expressed on two distinct cells. A bispecific antigen binding molecule as described herein can also form part of a multispecific antibody. The term “valent” as used within the current application denotes the presence of a specified number of binding sites specific for one distinct antigenic determinant in an antigen binding molecule that are specific for one distinct antigenic determinant. As such, the terms “bivalent”, “tetravalent”, and “hexavalent” denote the presence of two binding sites, four binding sites, and six binding sites specific for a certain antigenic determinant, respectively, in an antigen binding molecule. In particular aspects of the invention, the bispecific antigen binding molecules according to the invention can be monovalent for a certain antigenic determinant, meaning that they have only one binding site for said antigenic determinant or they can be bivalent or tetravalent for a certain antigenic determinant, meaning that they have two binding sites or four binding sites, respectively, for said antigenic determinant. The terms “full length antibody”, “intact antibody”, and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure. “Native antibodies” refer to naturally occurring immunoglobulin molecules with varying structures. For example, native IgG-class antibodies are heterotetrameric glycoproteins of about 150,000 daltons, composed of two light chains and two heavy chains thatare disulfide-bonded. From N- to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3), also called a heavy chain constant region. Similarly, from N- to C-terminus, each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a light chain constant domain (CL), also called a 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 divided into subtypes, e.g. 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. An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds 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, triabodies, tetrabodies, cross-Fab fragments; linear antibodies; single-chain antibody molecules (e.g. scFv); and single domain antibodies. For a review of certain antibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003). For a review of scFv fragments, see e.g. Plückthun, 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 discussion of Fab and F(ab')2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Patent No. 5,869,046. Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific, see, for example, EP 404,097; WO 1993 / 01161; Hudson et al., Nat Med 9, 129-134 (2003); and Hollinger et al., Proc Natl Acad Sci USA 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 comprising 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). Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g. E. coli or phage), as described herein. Papain digestion of intact antibodies produces two identical antigen-binding fragments, called “Fab” fragments containing each the heavy- and light-chain variable domains and also the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. As used herein, Thus, the term “Fab fragment” refers to an antibody fragment comprising a light chain fragment comprising a VL domain and a constant domain of a light chain (CL), and a VH domain and a first constant domain (CH1) of a heavy chain. 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 cysteins from the antibody hinge region. Fab’-SH are Fab’fragments wherein the cysteine residue(s) of the constant domains bear a free thiol group. Pepsin treatment yields an F(ab')2 fragment that has two antigen-combining sites (two Fab fragments) and a part of the Fc region. According to the present invention, the term “Fab fragment” also includes “crossFab fragments” or “crossover Fab fragments” as defined below. The term “crossFab fragment” or “xFab fragment” or “crossover Fab fragment” refers to a Fab fragment, wherein either the variable regions or the constant regions of the heavy and light chain are exchanged. Two different chain compositions of a crossover Fab molecule are possible and comprised in the bispecific antibodies of the invention: On the one hand, the variable regions of the Fab heavy and light chain are exchanged, i.e. the crossover Fab molecule 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 molecule is also referred to as CrossFab (VLVH). On the other hand, when the constant regions of the Fab heavy and light chain are exchanged, the crossover Fab molecule 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 crossoverFab molecule is also referred to as CrossFab (CLCH1).By "specific binding" is meant that the binding is selective for the antigen and can be discriminated from unwanted or non-specific interactions. The ability of an antigen binding molecule to bind to a specific antigen can be measured either through an enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to one of skill in the art, e.g. Surface Plasmon Resonance (SPR) technique (analyzed on a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)), and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, the extent of binding of an antigen binding molecule to an unrelated protein is less than about 10% of the binding of the antigen binding molecule to the antigen as measured, e.g. by SPR. In certain embodiments, an molecule that binds to the antigen has a dissociation constant (Kd) of ≤ 1 μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM, or ≤ 0.001 nM (e.g. 10-8M or less, e.g. from 10-8M to 10-13M, e.g. from 10-9M to 10-13M). “Affinity” or “binding affinity” refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g. an antibody) and its binding partner (e.g. an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g. antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd), which is the ratio of dissociation and association rate constants (koff and kon, respectively). Thus, equivalent affinities may comprise different rate constants, as long as the ratio of the rate constants remains the same. Affinity can be measured by common methods known in the art, including those described herein. A particular method for measuring affinity is Surface Plasmon Resonance (SPR).A “target cell antigen” as used herein refers to an antigenic determinant presented on the surface of a target cell, in particular a target cell in a tumor such as a cancer cell or a cell of the tumor stroma. Thus, the target cell antigen is a tumor-associated antigen. In particular, the “tumor-associated antigen” or TAA is Fibroblast Activation Protein (FAP). The term “Fibroblast activation protein (FAP)”, also known as Prolyl endopeptidase FAP or Seprase (EC 3.4.21), refers to any native FAP from any vertebrate source, including mammals such as primates (e.g. humans) non-human primates (e.g. cynomolgus monkeys) and rodents (e.g. mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed FAP as well as any form of FAP that results from processing in the cell. The term also encompasses naturally occurring variants of FAP, e.g., splice variants or allelic variants. In one embodiment, the antigen binding molecule of the invention is capable of specific binding to human, mouse and / or cynomolgus FAP. The amino acid sequence of human FAP is shown in UniProt (www.uniprot.org) accession no. Q12884 (version 149, SEQ ID NO:13), or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_004451.2. The extracellular domain (ECD) of human FAP extends from amino acid position 26 to 760. The amino acid sequence of an Avi-His-tagged human FAP is shown in SEQ ID NO: 14. The amino acid sequence of mouse FAP is shown in UniProt accession no. P97321 (version 126, SEQ ID NO:15), or NCBI RefSeq NP_032012.1. The extracellular domain (ECD) of mouse FAP extends from amino acid position 26 to 761. SEQ ID NO:16 shows the amino acid of a Avi-His-tagged mouse FAP. Preferably, an anti-FAP binding molecule of the invention binds to the extracellular domain of FAP. The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antigen binding molecule to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three complementary determining regions (CDRs). See, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. The term “complementarity determining region” or “CDR” as used herein refers to each of the regions of an antibody variable domain which are hypervariable in sequence and which determine antigen binding specificity. Generally, antibodies comprise six CDRs: three in the VH (CDR-H1, CDR-H2, CDR-H3), and three in the VL (CDR-L1, CDR-L2, CDR-L3). CDRs are defined by a variety of methods / systems by those skilled in the art. These systems and / or definitions have been developed and refined over a number of years and include Kabat, Chothia, IMGT, AbM, and Contact. The Kabat definition is based on sequence variability and generally is the most commonly used. The Chothia definition is based on the location of the structural loop regions. The IMGT system is based on sequence variability and location within the structure of the variable domain. The AbM definition is a compromise between Kabat and Chothia. The Contact definition is based on analyses of the available antibody crystal structures. Softwareprograms are available and known to those of skill in the art for analysis of antibody sequences and determination of CDRs. Unless otherwise indicated, the CDRs are determined according to Kabat et al., supra. One of skill in the art will understand that the CDR designations can also bedetermined according to Chothia, supra, McCallum, supra, or any other scientifically acceptednomenclature system. “Framework” or “FR” refers to variable domain residues other than complementary determining regions (CDRs). The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the CDR and FR sequences generally appear in the following sequence in VH (or VL): FR1-CDR-H1(CDR-L1)-FR2- CDR-H2(CDR-L2)- FR3- CDR-H3(CDR-L3)-FR4. 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 remainder of the heavy and / or light chain is derived from a different source or species. The “class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g. IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. 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. A “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non- human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may 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 undergone humanization. Other forms of "humanized antibodies" encompassed by the present invention are those in which the constant region has been additionally modified or changed from that of the original antibody to generate the properties according to the invention, especially in regard to C1q binding and / or Fc receptor (FcR) binding. The term "CH1 domain" denotes the part of an antibody heavy chain polypeptide that extends approximately from EU position 118 to EU position 215 (EU numbering system according to Kabat). In one aspect, a CH1 domain has the amino acid sequence of ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKV (SEQ ID NO: 17).The term "hinge region" denotes the part of an antibody heavy chain polypeptide that joins in a wild-type antibody heavy chain the CH1 domain and the CH2 domain, e. g. from about position 216 to about position 230 according to the EU number system of Kabat, or from about position 226 to about position 230 according to the EU number system of Kabat. The hinge regions of other IgG subclasses can be determined by aligning with the hinge-region cysteine residues of the IgG1 subclass sequence. The hinge region is normally a dimeric molecule consisting of two polypeptides with identical amino acid sequence. The hinge region generally comprises up to 25 amino acid residues and is flexible allowing the associated target binding sites to move independently. The hinge region can be subdivided into three domains: the upper, the middle, and the lower hinge domain (see e.g. Roux, et al., J. Immunol.161 (1998) 4083). In one aspect, the hinge region has the amino acid sequence DKTHTCPXCP (SEQ ID NO: 18), wherein X is either S or P. The term “Fc domain” or “Fc region” herein is used to define a C-terminal region of an antibody heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. An IgG Fc region comprises an IgG CH2 and an IgG CH3 domain. The “CH2 domain” of a human IgG Fc region usually extends from an amino acid residue at about position 231 to an amino acid residue at about position 340. (EU numbering system according to Kabat). In one aspect, a CH2 domain has the amino acidsequence of APELLGGPSV FLFPPKPKDT LMISRTPEVT CVWDVSHEDP EVKFNWYVDGVEVHNAKTKP REEQESTYRW SVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAK (SEQ ID NO: 19). The CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native Fc-region. It has been speculated that the carbohydrate may provide a substitute for the domain-domain pairing and help stabilize the CH2 domain. Burton, Mol. Immunol.22 (1985) 161-206. In one aspect, a carbohydrate chain is attached to the CH2 domain. The CH2 domain herein may be a native sequence CH2 domain or variant CH2 domain. The “CH3 domain” comprises the stretch of residues C-terminal to a CH2 domain in an Fc region (i.e. from an amino acid residue at about position 341 to an amino acid residue at about position 447 according to EU numbering system according to Kabat of an IgG). In one aspect,the CH3 domain has the amino acid sequence of GQPREPQVYT LPPSRDELTK NQVSLTCLVKGFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG (SEQ ID NO: 20). The CH3 region herein may be a native sequence CH3 domain or a variant CH3 domain (e.g. a CH3 domain with an introduced “protuberance” (“knob”) in one chain thereof and a corresponding introduced “cavity” (“hole”) in the other chain thereof; see US Patent No.5,821,333, expressly incorporated herein by reference). Such variant CH3 domains may be used to promote heterodimerization of two non- identical antibody heavy chains as herein described. In one embodiment, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavychain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, 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. The term “wild-type Fc domain” denotes an amino acid sequence identical to the amino acid sequence of an Fc domain found in nature. Wild-type human Fc domains include a native human IgG1 Fc-region (non-A and A allotypes), native human IgG2 Fc-region, native human IgG3 Fc-region, and native human IgG4 Fc-region as well as naturally occurring variants thereof. Wild-type Fc-regions are denoted in SEQ ID NO: 21 (IgG1, caucasian allotype), SEQ ID NO: 22 (IgG1, afroamerican allotype), SEQ ID NO: 23 (IgG2), SEQ ID NO:24 (IgG3) and SEQ ID NO:25 (IgG4). The term “variant (human) Fc domain” denotes an amino acid sequence which differs from that of a “wild-type” (human) Fc domain amino acid sequence by virtue of at least one “amino acid mutation”. In one aspect, the variant Fc-region has at least one amino acid mutation compared to a native Fc-region, e.g. from about one to about ten amino acid mutations, and in one aspect from about one to about five amino acid mutations in a native Fc- region. In one aspect, the (variant) Fc-region has at least about 95 % homology with a wild-type Fc-region. The “knob-into-hole” technology is described e.g. in US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Generally, the method involves introducing a protuberance (“knob”) at the interface of a first polypeptide and a corresponding cavity (“hole”) in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity so as to promote heterodimer formation and hinder homodimer formation. Protuberances are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g. tyrosine or tryptophan). Compensatory cavities of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). The protuberance and cavity can be made by altering the nucleic acid encoding the polypeptides, e.g. by site-specific mutagenesis, or by peptide synthesis. In a specific embodiment a knob modification comprises the amino acid substitution T366W in one of the two subunits of the Fc domain, and the hole modification comprises the amino acid substitutions T366S, L368A and Y407V in the other one of the two subunits of the Fc domain. In a further specific embodiment, the subunit of the Fc domain comprising the knob modification additionally comprises the amino acid substitution S354C, and the subunit of the Fc domain comprising the hole modification additionally comprises the amino acid substitution Y349C. Introduction of these two cysteine residues results in the formation of a disulfide bridge between the two subunits of the Fc region, thus further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).A "region equivalent to the Fc region of an immunoglobulin" is intended to include naturally occurring allelic variants of the Fc region of an immunoglobulin as well as variants having alterations which produce substitutions, additions, or deletions but which do not decrease substantially the ability of the immunoglobulin to mediate effector functions (such as antibody- dependent cellular cytotoxicity). For example, one or more amino acids can be deleted from the N-terminus or C-terminus of the Fc region of an immunoglobulin without substantial loss of biological function. Such variants can be selected according to general rules known in the art so as to have minimal effect on activity (see, e.g., Bowie, J. U. et al., Science 247:1306-10 (1990)). The term “effector function” refers to those biological activities attributable to the Fc region of an antibody, which vary with 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), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen presenting cells, down regulation of cell surface receptors (e.g. B cell receptor), and B cell activation. Fc receptor binding dependent effector functions can be mediated by the interaction of the Fc-region of an antibody with Fc receptors (FcRs), which are specialized cell surface receptors on hematopoietic cells. Fc receptors belong to the immunoglobulin superfamily, and have been shown to mediate both the removal of antibody-coated pathogens by phagocytosis of immune complexes, and the lysis of erythrocytes and various other cellular targets (e.g. tumor cells) coated with the corresponding antibody, via antibody dependent cell mediated cytotoxicity (ADCC) (see e.g. Van de Winkel, J.G. and Anderson, C.L., J. Leukoc. Biol.49 (1991) 511-524). FcRs are defined by their specificity for immunoglobulin isotypes: Fc receptors for IgG antibodies are referred to as FcγR. Fc receptor binding is described e.g. in Ravetch, J.V. and Kinet, J.P., Annu. Rev. Immunol.9 (1991) 457-492, Capel, P.J., et al., Immunomethods 4 (1994) 25-34; de Haas, M., et al., J. Lab. Clin. Med.126 (1995) 330-341; and Gessner, J.E., et al., Ann. Hematol.76 (1998) 231-248. Cross-linking of receptors for the Fc-region of IgG antibodies (FcγR) triggers a wide variety of effector functions including phagocytosis, antibody-dependent cellular cytotoxicity, and release of inflammatory mediators, as well as immune complex clearance and regulation of antibody production. In humans, three classes of FcγR have been characterized, which are: - Fc^RI (CD64) binds monomeric IgG with high affinity and is expressed on macrophages, monocytes, neutrophils and eosinophils. Modification in the Fc-region IgG at least at one of the amino acid residues E233-G236, P238, D265, N297, A327 and P329 (numbering according to EU index of Kabat) reduce binding to FcγRI. IgG2 residues at positions 233–236, substituted into IgG1 and IgG4, reduced binding to FcγRI by 10³-fold and eliminated the human monocyte response to antibody-sensitized red blood cells (Armour, K.L., et al., Eur. J. Immunol.29 (1999) 2613–2624).-Fc^RII (CD32) binds complexed IgG with medium to low affinity and is widely expressed. This receptor can be divided into two sub-types, Fc^RIIA and Fc^RIIB. Fc^RIIA is found on many cells involved in killing (e.g. macrophages, monocytes, neutrophils) and seems able to activate the killing process. FcγRIIB seems to play a role in inhibitory processes and is found on B cells, macrophages and on mast cells and eosinophils. On B-cells it seems to function to suppress further immunoglobulin production and isotype switching to, for example, the IgE class. On macrophages, FcγRIIB acts to inhibit phagocytosis as mediated through FcγRIIA. On eosinophils and mast cells the B-form may help to suppress activation of these cells through IgE binding to its separate receptor. Reduced binding for FcγRIIA is found e.g. for antibodies comprising an IgG Fc-region with mutations at least at one of the amino acid residues E233- G236, P238, D265, N297, A327, P329, D270, Q295, A327, R292, and K414 (numbering according to EU index of Kabat). - Fc^RIII (CD16) binds IgG with medium to low affinity and exists as two types. Fc^RIIIA is found on NK cells, macrophages, eosinophils and some monocytes and T cells and mediates ADCC. FcγRIIIB is highly expressed on neutrophils. Reduced binding to Fc^RIIIA is found e.g. for antibodies comprising an IgG Fc-region with mutation at least at one of the amino acid residues E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, S239, E269, E293, Y296, V303, A327, K338 and D376 (numbering according to EU index of Kabat). Mapping of the binding sites on human IgG1 for Fc receptors, the above mentioned mutation sites and methods for measuring binding to FcγRI and FcγRIIA are described in Shields, R.L., et al. J. Biol. Chem.276 (2001) 6591-6604. The term “ADCC” or “antibody-dependent cellular cytotoxicity” is a function mediated by Fc receptor binding and refers to lysis of target cells by an antibody as reported herein in the presence of effector cells. The capacity of the antibody to induce the initial steps mediating ADCC is investigated by measuring their binding to Fcγ receptors expressing cells, such as cells, recombinantly expressing FcγRI and / or FcγRIIA or NK cells (expressing essentially FcγRIIIA). In particular, binding to FcγR on NK cells is measured. An “activating Fc receptor” is an Fc receptor that following engagement by an Fc region of an antibody elicits signaling events that stimulate the receptor-bearing cell to perform effector functions. Activating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89). A particular activating Fc receptor is human FcγRIIIa (see UniProt accession no. P08637, version 141). The term “CD40”, as used herein, refers to any native CD40 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 CD40 as well as any form of CD40 that results from processing in the cell. The term also encompasses naturally occurring variants of CD40, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human CD40 is shown in SEQ ID NO:26 (Uniprot P25942, version 200) and theamino acid sequence of an exemplary mouse CD40 is shown in SEQ ID NO: 27 (Uniprot P27512, version 160). The CD40 antigen is a 50 kDa cell surface glycoprotein which belongs to the Tumor Necrosis Factor Receptor (TNF-R) family. (Stamenkovic et al. (1989), EMBO J.8: 1403-10). CD40 is expressed in many normal and tumor cell types, including B lymphocytes, dendritic cells, monocytes, macrophages, thymus epithelium, endothelial cells, fibroblasts, and smooth muscle cells. CD40 is expressed in all B-lymphomas and in 70% of all solid tumors and is up-regulated in antigen presenting cells (APCs) by maturation signals, such as IFN-gamma and GM-CSF. CD40 activation also induces differentiation of monocytes into functional dendritic cells (DCs) and enhances cytolytic activity of NK cells through APC-CD40 induced cytokines. Thus CD40 plays an essential role in the initiation and enhancement of immune responses by inducing maturation of APCs, secretion of helper cytokines, upregulation of costimulatory molecules, and enhancement of effector functions. The term "CD40 agonist" as used herein includes any moiety that agonizes the CD40 / CD40L interaction. CD40 as used in this context refers preferably to human CD40, thus the CD40 agonist is preferably an agonist of human CD40. Typically, the moiety will be an agonistic CD40 antibody or antibody fragment. The terms “anti-CD40 antibody”, “anti-CD40”, “CD40 antibody and “an antibody that specifically binds to CD40” refer to an antibody that is capable of binding CD40 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting CD40. In one aspect, the extent of binding of an anti-CD40 antibody to an unrelated, non-CD40 protein is less than about 10% of the binding of the antibody to CD40 as measured, e.g., by a radioimmunoassay (RIA) or flow cytometry (FACS). In certain embodiments, an antibody that binds to CD40 has a dissociation constant (KD) of ≤ 1μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM, or ≤ 0.001 nM (e.g.10-6M or less, e.g. from 10-68M to 10-13M, e.g., from 10-8M to 10-10M). The term “peptide linker” refers to a peptide comprising one or more amino acids, typically about 2 to 20 amino acids. Peptide linkers are known in the art or are described herein. Suitable, non-immunogenic linker peptides are, for example, (G4S)n, (SG4)n or G4(SG4)n peptide linkers, wherein “n” is generally a number between 1 and 10, typically between 2 and 4. The term ”amino acid” as used within this application denotes the group of naturally occurring carboxy α-amino acids comprising alanine (three letter code: ala, one letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine (cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V). By “fused” or “connected” is meant that the components (e.g. a heavy chain of an antibody and a Fab fragment) are linked by peptide bonds, either directly or via one or more peptide linkers.In certain aspects, amino acid sequence variants of the bispecific antigen bindingmolecules provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antigen binding molecules. Amino acid sequence variants of the bispecific antigen binding molecules may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the molecules, or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into and / or substitutions of residues within the amino acid sequences of the bispecific antigen binding molecules. Any combination of deletion, insertion, and substitution can be made to arrive at the final antigen binding molecule, provided that the final antigen binding molecule possesses the desired characteristics, e.g., antigen-binding. Sites of interest for substitutional mutagenesis include the CDRs and Framework (FRs). Conservative substitutions are provided in Table C under the heading “Preferred Substitutions” and further described below in reference to amino acid side chain classes (1) to (6). Amino acid substitutions may be introduced into the molecule of interest and the products screened for a desired activity, e.g., retained / improved antigen binding, decreased immunogenicity, or improved ADCC or CDC. An "effective amount" of an agent refers to the amount that is necessary to result in a physiological change in the cell or tissue to which it is administered. The combination therapies in accordance with the invention have a synergistic effect. A "synergistic effect" of two compounds is one in which the effect of the combination of the two agents is greater than the sum of their individual effects and is statistically different from the controls and the single drugs. In another embodiment, the combination therapies disclosed herein have an additive effect. An "additive effect" of two compounds is one in which the effect of the combination of the two agents is the sum of their individual effects and is statistically different from either the controls and / or the single drugs. A "therapeutically effective amount" of an agent, e.g. a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. A therapeutically effective amount of an agent for example eliminates, decreases, delays, minimizes or prevents adverse effects of a disease. An “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g. cows, sheep, cats, dogs, and horses), primates (e.g. humans and non- human primates such as monkeys), rabbits, and rodents (e.g. mice and rats). Particularly, the individual or subject is a human. The term "pharmaceutical composition" refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical composition, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable excipient includes, but is not limited to, a buffer, a stabilizer, or a preservative. The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or warnings concerning the use of such therapeutic products. 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 the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, the molecules of the invention are used to delay development of a disease or to slow the progression of a disease. The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth, i.e proliferative diseases, such as solid tumors or melanoma. A "tumor" comprises one or more cancerous cells. The term “solid tumor” as used herein refers to an abnormal mass of tissue that usually does not contain cysts or liquid areas. Solid tumors may be benign or malignant. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include breast cancer, squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer including small- cell lung cancer, non- small cell lung cancer ("NSCLC"), adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head and neck cancer. In one particular aspect, the cancer is a “FAP-expressing cancer”. Tumor types known to express FAP include non small cell lung cancer (NSCLC), small cell lung cancer (SCLC), triple negative breast cancer (TNBC), cutaneous melanoma, urothelial cancer, mesothelioma, hepatocellular carcinoma (HCC), head and neck squamous cell carcinoma (HNSCC), esophageal squamous cell carcinoma (ESCC), and cervical squamous cell carcinoma. Further FAP- expressing cancers are pancreatic cancer, gastric cancer and ovarian cancer.Reference to a tumor or cancer as a "Stage 0," "Stage I," "Stage II," "Stage III," or "Stage IV", and various sub-stages within this classification, indicates classification of the tumor or cancer using the Overall Stage Grouping or Roman Numeral Staging methods known in the art. Although the actual stage of the cancer is dependent on the type of cancer, in general, a Stage 0 cancer is an in situ lesion, a Stage I cancer is small localized tumor, a Stage II and III cancer is a local advanced tumor which exhibits involvement of the local lymph nodes, and a Stage IV cancer represents metastatic cancer. The specific stages for each type of tumor is known to the skilled clinician. An "advanced" cancer is one which has spread outside the site or organ of origin, either by local invasion or metastasis. Accordingly, the term "advanced" cancer includes both locally advanced and metastatic disease. A "refractory" cancer is one which progresses even though an anti-tumor agent, such as a chemotherapy, is being administered to the cancer patient. An example of a refractory cancer is one which is platinum refractory. A "recurrent" cancer is one which has regrown, either at the initial site or at a distant site, after a response to initial therapy, such as surgery. A "locally recurrent" cancer is cancer that returns after treatment in the same place as a previously treated cancer. An “operable" or "resectable" cancer is cancer which is confined to the primary organ and suitable for surgery (resection). A "non-resectable" or "unresectable" cancer is not able to be removed (resected) by surgery. Preferably, a "patient" or "subject" is a human patient. The patient may be a "cancer patient," i.e. one who is suffering from one or more solid tumors. A "patient population" refers to a group of cancer patients. Such populations can be used to demonstrate statistically significant efficacy and / or safety of a drug, such as Pertuzumab. A "relapsed" patient is one who has signs or symptoms of cancer after remission. Optionally, the patient has relapsed after adjuvant or neoadjuvant therapy. The terms "combination therapy" or "combined treatment" or "in combination" as used herein denote any form of concurrent or parallel treatment with at least two distinct therapeutic agents. "Neoadjuvant therapy" or "preoperative therapy" herein refers to therapy given prior to surgery. The goal of neoadjuvant therapy is to provide immediate systemic treatment, potentially eradicating micrometastases that would otherwise proliferate if the standard sequence of surgery followed by systemic therapy were followed. Neoadjuvant therapy may also help to reduce tumor size thereby allowing complete resection of initially unresectable tumors or preserving portions of the organ and its functions. Furthermore, neoadjuvant therapy permits an in vivo assessment of drug efficacy, which may guide the choice of subsequent treatments. “Adjuvant therapy” herein refers to therapy given after definitive surgery, where no evidence of residual disease can be detected, so as to reduce the risk of disease recurrence. The goal of adjuvant therapy is to prevent recurrence of the cancer, and therefore to reduce thechance of cancer-related death. Adjuvant therapy herein specifically excludes neoadjuvant therapy. “Chemotherapy” refers to the use of a chemotherapeutic agent useful in the treatment of cancer. A “chemotherapeutic agent” is a chemical compound useful in the treatment of cancer, regardless of mechanism of action. Classes of chemotherapeutic agents include, but are not limited to: alkylating agents, antimetabolites, spindle poison plant alkaloids, cytotoxic / antitumor antibiotics, topoisomerase inhibitors, antibodies, photosensitizers, and kinase inhibitors. The term “checkpoint inhibitors” refers to a type of immunotherapy agents that block immune checkpoint proteins, which are key regulators of the immune system. These proteins can prevent the immune system from attacking tumor cells by sending “off” signals to T cells. By blocking checkpoint proteins such as CTLA-4, PD-1, PD-L1 and LAG-3, checkpoint inhibitors prevent these “off” signals. The term "agent blocking PD-L1 / PD-1 interaction" is a molecule that inhibits the interaction of a PD-1 axis binding partner with either one or more of its binding partner, so as to remove T-cell dysfunction resulting from signaling on the PD- 1 signaling axis - with a result being to restore or enhance T-cell function, e.g., proliferation, cytokine production, target cell killing. As used herein, the term “agent blocking PD-L1 / PD-1 interaction” includes an anti-PD- L1 antibody or an anti-PD1 antibody. The terms “anti-PD-L1 antibody” and “an antibody that binds to PD-L1” refer to an antibody that is capable of binding PD-L1 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting PD-L1. In one aspect, the anti-PD-L1 antibody is atezolizumab or durvalumab, preferably atezolizumab. The terms “anti-PD-1 antibody” and “an antibody that binds to PD-1” refer to an antibody that is capable of binding PD-1 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting PD-1. In one aspect, the anti-PD-1 antibody is pembrolizumab or nivolumab, preferably pembrolizumab. The term "detection" includes any means of detecting, including direct and indirect detection. As used herein, the term "reagent that specifically detects expression levels" refers to reagents used to detect the expression of one or more genes (e.g., including but not limited to, the cancer markers of the present invention). Examples of suitable reagents include but are not limited to, nucleic acid probes capable of specifically hybridizing to the gene of interest, aptamers, PCR primers capable of specifically amplifying the gene of interest, and antibodies capable of specifically binding to proteins expressed by the gene of interest. The term “biomarker” as used herein refers to an indicator, e.g., predictive, diagnostic, and / or prognostic, which can be detected in a sample. The biomarker may serve as an indicator of a particular subtype of a disease or disorder (e.g., cancer) characterized by certain, molecular,pathological, histological, and / or clinical features. In some aspects, a biomarker is a gene. Biomarkers include, but are not limited to, polynucleotides (e.g., DNA, and / or RNA), polynucleotide copy number alterations (e.g., DNA copy numbers), polypeptides, polypeptide and polynucleotide modifications (e.g. posttranslational modifications), carbohydrates, and / or glycolipid-based molecular markers. The terms “biomarker signature,” “signature,” “biomarker expression signature,” or “expression signature” are used interchangeably herein and refer to one or a combination of biomarkers whose expression is an indicator, e.g., predictive, diagnostic, and / or prognostic. The biomarker signature may serve as an indicator of a particular subtype of a disease or disorder (e.g., cancer) characterized by certain molecular, pathological, histological, and / or clinical features. In some aspects, the biomarker signature is a “gene signature.” The term “gene signature” is used interchangeably with “gene expression signature” and refers to one or a combination of polynucleotides whose expression is an indicator, e.g., predictive, diagnostic, and / or prognostic. In some embodiments, the biomarker signature is a “protein signature.” The term “protein signature” is used interchangeably with “protein expression signature” and refers to one or a combination of polypeptides whose expression is an indicator, e.g., predictive, diagnostic, and / or prognostic. The "amount" or "level" of a biomarker associated with an increased clinical benefit to an individual is a detectable level in a biological sample. These can be measured by methods known to one skilled in the art and also disclosed herein. The expression level or amount of biomarker assessed can be used to determine the response to the treatment. The terms "level of expression" or "expression level" in general are used interchangeably and generally refer to the amount of a biomarker in a biological sample. "Expression" generally refers to the process by which information (e.g., gene-encoded and / or epigenetic) is converted into the structures present and operating in the cell. Therefore, as used herein, "expression" may refer to transcription into a polynucleotide, translation into a polypeptide, or even polynucleotide and / or polypeptide modifications (e.g., posttranslational modification of a polypeptide). Fragments of the transcribed polynucleotide, the translated polypeptide, or polynucleotide and / or polypeptide modifications (e.g., posttranslational modification of a polypeptide) shall also be regarded as expressed whether they originate from a transcript generated by alternative splicing or a degraded transcript, or from a post-translational processing of the polypeptide, e.g., by proteolysis. "Expressed genes" include those that are transcribed into a polynucleotide as mRNA and then translated into a polypeptide, and also those that are transcribed into RNA but not translated into a polypeptide (for example, transfer and ribosomal RNAs). Gene expression can be regulated at many stages in the process. "Up-regulation" or "activation" refers to regulation that increases the production of gene expression products (e.g., RNA or protein), while "down- regulation" or "repression" refers to regulation that decrease production. Molecules (e.g.,transcription factors) that are involved in up-regulation or down-regulation are often called "activators" and "repressors," respectively. The term “reference level” herein refers to a predetermined value. As a skilled person will appreciate the reference level is predetermined and set to meet the requirements in terms of e.g. specificity and / or sensitivity. These requirements can vary, e.g. from regulatory body to regulatory body. It may for example be that assay sensitivity or specificity, respectively, has to be set to certain limits, e.g.80%, 90% or 95%. These requirements may also be defined in terms of positive or negative predictive values. Nonetheless, based on the teaching given in the present invention it will always be possible to arrive at the reference level meeting those requirements. In one embodiment the reference level is determined in healthy individuals. The reference value in one embodiment has been predetermined in the disease entity to which the patient belongs. In certain embodiments the reference level can e.g. be set to any percentage between 25% and 75% of the overall distribution of the values in a disease entity investigated. In other aspects, the reference level can e.g. be set to the median, tertiles or quartiles as determined from the overall distribution of the values in a disease entity investigated. In one aspect, the reference level is set to the median value as determined from the overall distribution of the values in a disease entity investigated. In certain aspects, the term “increase”, “increased” or “above” refers to a level above the reference level. “Amplification,” as used herein generally refers to the process of producing multiple copies of a desired sequence. “Multiple copies” mean at least two copies. A “copy” does not necessarily mean perfect sequence complementarity or identity to the template sequence. For example, copies can include nucleotide analogs such as deoxyinosine, intentional sequence alterations (such as sequence alterations introduced through a primer comprising a sequence that is hybridizable, but not complementary, to the template), and / or sequence errors that occur during amplification. The term “multiplex-PCR” refers to a single PCR reaction carried out on nucleic acid obtained from a single source (e.g., an individual) using more than one primer set for the purpose of amplifying two or more DNA sequences in a single reaction. "Stringency" of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature which can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995). "Stringent conditions" or "high stringency conditions", as defined herein, can be identified by those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate at 50°C; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v / v) formamide with 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 50mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42°C; or (3) overnight hybridization in a solution that employs 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt’s solution, sonicated salmon sperm DNA (50 μg / ml), 0.1% SDS, and 10% dextran sulfate at 42°C, with a 10 minute wash at 42°C in 0.2 x SSC (sodium chloride / sodium citrate) followed by a 10 minute high-stringency wash consisting of 0.1 x SSC containing EDTA at 55°C. "Moderately stringent conditions" can be identified as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength and %SDS) less stringent that those described above. An example of moderately stringent conditions is overnight incubation at 37°C in a solution comprising: 20% formamide, 5 x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 x Denhardt’s solution, 10% dextran sulfate, and 20 mg / ml denatured sheared salmon sperm DNA, followed by washing the filters in 1 x SSC at about 37-50°C. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like. The technique of “polymerase chain reaction” or “PCR” as used herein generally refers to a procedure wherein minute amounts of a specific piece of nucleic acid, RNA and / or DNA, are amplified as described in U.S. Pat. No.4,683,195 issued 28 July 1987. Generally, sequence information from the ends of the region of interest or beyond needs to be available, such that oligonucleotide primers can be designed; these primers will be identical or similar in sequence to opposite strands of the template to be amplified. The 5' terminal nucleotides of the two primers may coincide with the ends of the amplified material. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA transcribed from total cellular RNA, bacteriophage or plasmid sequences, etc. See generally Mullis et al., Cold Spring Harbor Symp. Quant. Biol., 51: 263 (1987); Erlich, ed., PCR Technology, (Stockton Press, NY, 1989). As used herein, PCR is considered to be one, but not the only, example of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample, comprising the use of a known nucleic acid (DNA or RNA) as a primer and utilizes a nucleic acid polymerase to amplifyor generate a specific piece of nucleic acid or to amplify or generate a specific piece of nucleic acid which is complementary to a particular nucleic acid. “Quantitative real time polymerase chain reaction” or “qRT-PCR” refers to a form of PCR wherein the amount of PCR product is measured at each step in a PCR reaction. This technique has been described in various publications including Cronin et al., Am. J. Pathol. 164(1):35-42 (2004); and Ma et al., Cancer Cell 5:607-616 (2004). The term "microarray" refers to an ordered arrangement of hybridizable array elements, preferably polynucleotide probes, on a substrate. The term "polynucleotide," when used in singular or plural, generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. Thus, for instance, polynucleotides as defined herein include, without limitation, single- and double-stranded DNA, DNA including single- and double-stranded regions, single- and double-stranded RNA, and RNA including single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or include single- and double-stranded regions. In addition, the term "polynucleotide" as used herein refers to triple- stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a triple-helical region often is an oligonucleotide. The term "polynucleotide" specifically includes cDNAs. The term includes DNAs (including cDNAs) and RNAs that contain one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotides" as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritiated bases, are included within the term "polynucleotides" as defined herein. In general, the term "polynucleotide" embraces all chemically, enzymatically and / or metabolically modified forms of unmodified polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells. The term "oligonucleotide" refers to a relatively short polynucleotide, including, without limitation, single-stranded deoxyribonucleotides, single- or double-stranded ribonucleotides, RNA:DNA hybrids and double- stranded DNAs. Oligonucleotides, such as single- stranded DNA probe oligonucleotides, are often synthesized by chemical methods, for example using automated oligonucleotide synthesizers that are commercially available. However,oligonucleotides can be made by a variety of other methods, including in vitro recombinantDNA-mediated techniques and by expression of DNAs in cells and organisms.The term "diagnosis" is used herein to refer to the identification or classification of a molecular or pathological state, disease or condition (e.g., cancer). For example, “diagnosis” may refer to identification of a particular type of cancer. “Diagnosis” may also refer to the classification of a particular subtype of cancer, e.g., by histopathological criteria, or by molecular features (e.g., a subtype characterized by expression of one or a combination of biomarkers (e.g., particular genes or proteins encoded by said genes)). The term “immunofluorescence (IF) analysis” refers to a light microscopy-based method that allows for the detection and localization of target proteins within a cell or tissue sample. It utilizes fluorophore-conjugated antibodies to visualize the position of target proteins by exciting the fluorophore, which emits light at a specific wavelength observed through a fluorescence microscope. Advanced IF techniques enable simultaneous detection of multiple targets in a so- called multiplex IF panel. “RNA sequencing” (RNA-Seq) is a technique that utilizes next-generation sequencing (NGS) technologies to analyze the presence and quantity of RNA molecules in a biological sample. This method provides a comprehensive snapshot of gene expression, known as the transcriptome, within the sample. RNA-Seq is capable of examining various RNA populations, including mRNA, rRNA, tRNA, and small RNAs like miRNA. It offers insights into alternative splicing, post-transcriptional modifications, gene fusions, mutations, and changes in gene expression levels under different conditions or treatments. The term “sample,” as used herein, refers to a composition that is obtained or derived from a subject and / or individual of interest that contains a cellular and / or other molecular entity that is to be characterized and / or identified, for example based on physical, biochemical, chemical and / or physiological characteristics. For example, the phrase “disease sample” and variations thereof refers to any sample obtained from a subject of interest that would be expected or is known to contain the cellular and / or molecular entity that is to be characterized. Samples include, but are not limited to, primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous fluid, lymph fluid, synovial fluid, follicular fluid, seminal fluid, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebro-spinal fluid, saliva, sputum, tears, perspiration, mucus, tumor lysates, and tissue culture medium, tissue extracts such as homogenized tissue, tumor tissue, cellular extracts, and combinations thereof. By "tissue sample” or “cell sample" is meant a collection of similar cells obtained from a tissue of a subject or individual. The source of the tissue or cell sample may be solid tissue as from a fresh, frozen and / or preserved organ, tissue sample, biopsy, and / or aspirate; blood or anyblood constituents such as plasma; bodily fluids such as cerebral spinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; cells from any time in gestation or development of the subject. The tissue sample may also be primary or cultured cells or cell lines. Optionally, the tissue or cell sample is obtained from a disease tissue / organ. The tissue sample may contain compounds which are not naturally intermixed with the tissue in nature such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, or the like. A “reference sample”, “reference cell”, “reference tissue”, “control sample”, “control cell”, or “control tissue”, as used herein, refers to a sample, cell, tissue, standard, or level that is used for comparison purposes. In one embodiment, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy and / or non- diseased part of the body (e.g., tissue or cells) of the same subject or individual. For example, healthy and / or non-diseased cells or tissue adjacent to the diseased cells or tissue (e.g., cells or tissue adjacent to a tumor). In another embodiment, a reference sample is obtained from an untreated tissue and / or cell of the body of the same subject or individual. In yet another embodiment, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy and / or non-diseased part of the body (e.g., tissues or cells) of an individual who is not the subject or individual. In even another embodiment, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from an untreated tissue and / or cell of the body of an individual who is not the subject or individual. For the purposes herein a “section” of a tissue sample is meant a single part or piece of a tissue sample, e.g. a thin slice of tissue or cells cut from a tissue sample. It is understood that multiple sections of tissue samples may be taken and subjected to analysis, provided that it is understood that the same section of tissue sample may be analyzed at both morphological and molecular levels, or analyzed with respect to both polypeptides and polynucleotides. By “correlate” or “correlating” is meant comparing, in any way, the performance and / or results of a first analysis or protocol with the performance and / or results of a second analysis or protocol. For example, one may use the results of a first analysis or protocol in carrying out a second protocols and / or one may use the results of a first analysis or protocol to determine whether a second analysis or protocol should be performed. With respect to the aspect of polypeptide analysis or protocol, one may use the results of the polypeptide expression analysis or protocol to determine whether a specific therapeutic regimen should be performed. With respect to the aspect of polynucleotide analysis or protocol, one may use the results of thepolynucleotide expression analysis or protocol to determine whether a specific therapeutic regimen should be performed. "Individual response" or “response” can be assessed using any endpoint indicating a benefit to the individual, including, without limitation, (1) inhibition, to some extent, of disease progression (e.g., cancer progression), including slowing down and complete arrest; (2) a reduction in tumor size; (3) inhibition (i.e., reduction, slowing down or complete stopping) of cancer cell infiltration into adjacent peripheral organs and / or tissues; (4) inhibition (i.e. reduction, slowing down or complete stopping) of metatasis; (5) relief, to some extent, of one or more symptoms associated with the disease or disorder (e.g., cancer); (6) increase or extend in the length of survival, including overall survival and progression free survival; and / or (7) decreased mortality at a given point of time following treatment. An "effective response" of a patient or a patient's "responsiveness" to treatment with a medicament and similar wording refers to the clinical or therapeutic benefit imparted to a patient at risk for, or suffering from, a disease or disorder, such as cancer. In one embodiment, such benefit includes any one or more of: extending survival (including overall survival and progression free survival); resulting in an objective response (including a complete response or a partial response); or improving signs or symptoms of cancer. In one embodiment, the presence of the biomarker is used to identify a patient who is more likely to respond to treatment with a medicament, relative to a patient that does not have the presence of the biomarker. In another embodiment, the presence of the biomarker is used to determine that a patient will have an increase likelihood of benefit from treatment with a medicament, relative to a patient that does not have the presence of the biomarker. An "effective amount" of an agent refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. A “therapeutically effective amount” refers to an amount of a therapeutic agent to treat or prevent a disease or disorder in a mammal. In the case of cancers, the therapeutically effective amount of the therapeutic agent may reduce the number of cancer cells; reduce the primary tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and / or relieve to some extent one or more of the symptoms associated with the disorder. To the extent the drug may prevent growth and / or killexisting cancer cells, it may be cytostatic and / or cytotoxic. For cancer therapy, efficacy in vivocan, for example, be measured by assessing the duration of survival, time to disease progression (TTP), the response rates (RR), duration of response, and / or quality of life. It is understood that the singular form "a", "an", and "the" includes plural references unless indicated otherwise. Exemplary FAP-targeted CD40 agonistic antibodies for use in the invention Provided herein are bispecific agonistic CD40 antibodies comprising at least one antigen binding domain capable of specific binding to Fibroblast Activation Protein (FAP) (FAP-CD40 antibodies) for use in treating cancer in combination with at least one anti-cancer agent, wherein the bispecific FAP-CD40 antibodies increase the density of intratumoral DC-LAMP+ DCs and modulates the immune system in the tumor microenvironment so that it is more sensitive to treatment with the anti-cancer agent, meaning that the treatment with the anti-cancer agent can be more effective. In one aspect, provided is a bispecific FAP-CD40 antibody that comprises at least one antigen binding domain capable of specific binding to CD40 comprising a heavy chain variable region VH (CD40) comprising the amino acid sequence of SEQ ID NO:1 and a light chain variable region VL (CD40) comprising the amino acid sequence of SEQ ID NO:2, and at least one antigen binding domain capable of specific binding to Fibroblast Activation Protein (FAP) comprising (a) a VH (FAP) comprising the amino acid sequence of SEQ ID NO:3 and a VL (FAP) comprising the amino acid sequence of SEQ ID NO:4, (b) a VH (FAP) comprising the amino acid sequence of SEQ ID NO:5 and a VL (FAP) comprising the amino acid sequence of SEQ ID NO:6, or (c) a VH (FAP) comprising the amino acid sequence of SEQ ID NO:7 and a light chain variable region VL (FAP) comprising the amino acid sequence of SEQ ID NO:8. In particular aspects, the bispecific agonistic FAP-CD40 antibody comprises at least one antigen binding domain capable of specific binding to FAP comprising a heavy chain variable region VH (FAP) comprising the amino acid sequence of SEQ ID NO:3 and a light chain variable region VL (FAP) comprising the amino acid sequence of SEQ ID NO:4. Thus, in particular aspects, the bispecific agonistic FAP-CD40 antibody comprises a heavy chain variable region VH (CD40) comprising the amino acid sequence of SEQ ID NO:1 and a light chain variable region VL (CD40) comprising the amino acid sequence of SEQ ID NO:2, and at least one antigen binding domain capable of specific binding to FAP comprising a heavy chain variable region VH (FAP) comprising the amino acid sequence of SEQ ID NO:3 and a light chain variable region VL (FAP) comprising the amino acid sequence of SEQ ID NO:4.In another aspects, the bispecific agonistic CD40-antigen binding molecule comprises (i) at least one antigen binding domain capable of specific binding to CD40, comprising a heavy chain variable region VH (CD40) comprising the amino acid sequence of SEQ ID NO:1 and a light chain variable region VL (CD40) comprising the amino acid sequence of SEQ ID NO:2, and (ii) at least one antigen binding domain capable of specific binding to FAP, comprising a heavy chain variable region VH (FAP) comprising an amino acid sequence of SEQ ID NO:5 and a light chain variable region VL (FAP) comprising an amino acid sequence of SEQ ID NO:6. In other aspects, the bispecific agonistic CD40-antigen binding molecule comprises (i) at least one antigen binding domain capable of specific binding to CD40, comprising a heavy chain variable region VH (CD40) comprising the amino acid sequence of SEQ ID NO:1 and a light chain variable region VL (CD40) comprising the amino acid sequence of SEQ ID NO:2, and (ii) at least one antigen binding domain capable of specific binding to FAP, comprising a heavy chain variable region VH (FAP) comprising an amino acid sequence of SEQ ID NO:7 and a light chain variable region VL (FAP) comprising an amino acid sequence of SEQ ID NO:8. In one aspect of the invention, the bispecific FAP-CD40 antibody comprises a Fc domain of human IgG1 subclass with the amino acid mutations L234A, L235A and P329G (numbering according to Kabat EU index). Alternatively, it may comprise a Fc domain of human IgG1 or IgG4 subclass with one or more amino acid substitutions that reduce the binding affinity of the antibody to an Fc receptor and / or effector function. In some aspects, the bispecific FAP-CD40 antibody comprises a) at least two Fab fragments capable of specific binding to CD40 fused at its C-terminus to the N-terminus of a Fc region, and (b) one antigen binding domain capable of specific binding to FAP fused at its N-terminus to the C-terminus of the Fc region. In some aspects, the bispecific FAP-CD40 antibody comprises a) at least two Fab fragments capable of specific binding to CD40 fused at its C-terminus to the N-terminus of a Fc region, and (b) a cross-fab fragment capable of specific binding to FAP fused to the C-terminus of the Fc region. In particular aspects, the bispecific FAP-CD40 antibody comprises a cross-fab fragment capable of specific binding to FAP, wherein the VH-Ckappa chain of the cross-fab fragment is fused to the C-terminus of the Fc region. In particular aspects, the bispecific agonistic CD40- antigen binding molecule comprises two Fab fragments capable of specific binding to CD40 fused at its C-terminus to the N-terminus of a Fc region. In particular aspects, the bispecific agonistic CD40-antigen binding molecule is characterized by bivalent binding to CD40 and monovalent binding to FAP.In one aspect, the bispecific FAP-CD40 antibody comprises (a) two light chains and two heavy chains of an antibody comprising two Fab fragments capable of specific binding to CD40 and the Fc domain, and (b) a VH and VL domain capable of specific binding to a target cell antigen, wherein the VH domain and the VL domain are each connected via a peptide linker to one of the C-termini of the two heavy chains. In a further aspect, provided is a bispecific FAP-CD40 antibody, comprising (a) two heavy chains, each heavy chain comprising a VH and CH1 domain of a Fab fragment capable of specific binding to CD40 and a Fc region subunit, (b) two light chains, each light chain comprising a VL and CL domain of a Fab fragment capable of specific binding to CD40, and (c) a cross-fab fragment capable of specific binding to FAP comprising a VL-CH1 chain and a VH-CL chain, wherein the VH-CL chain is connected to the C-terminus of one of the two heavy chains of (a). In one aspect, the VH-CL (VH-Ckappa) chain is connected to the C-terminus of the Fc knob heavy chain. In one particular aspect, provided is a bispecific FAP-CD40 antibody comprising two light chains, each comprising the amino acid sequence of SEQ ID NO:9, one light chain comprising the amino acid sequence of SEQ ID NO:10, a first heavy chain comprising the amino acid sequence of SEQ ID NO:11, and a second heavy chain comprising the amino acid sequence of SEQ ID NO:12. This bispecific FAP-CD40 antibody is also described herein as RO7300490. In another aspect, the bispecific FAP-CD40 antibody is characterized by tetravalent or trivalent binding to CD40 and monovalent binding to FAP. In some aspects, the bispecific FAP- CD40 antibody is characterized by tetravalent binding to CD40 and monovalent binding to FAP. In some aspects, the bispecific FAP-CD40 antibody comprises four Fab fragments capable of specific binding to CD40, wherein each two Fab fragments are fused to each other and fused at its C-terminus to the N-terminus of a Fc region. In another aspect, provided are murine surrogates of bispecific FAP-CD40 antibodies. In one aspect, provided is a bispecific FAP-CD40 antibody comprising two light chains, each comprising the amino acid sequence of SEQ ID NO:9, one light chain comprising the amino acid sequence of SEQ ID NO:28, a first heavy chain comprising the amino acid sequence of SEQ ID NO:29, and a second heavy chain comprising the amino acid sequence of SEQ ID NO:12 (P1AE2302). In another aspect, provided is a bispecific FAP-CD40 antibody comprisingtwo light chains, each comprising the amino acid sequence of SEQ ID NO:30, one light chain comprising the amino acid sequence of SEQ ID NO:31, a first heavy chain comprising the amino acid sequence of SEQ ID NO:32, and a second heavy chain comprising the amino acid sequence of SEQ ID NO:33 (P1AE5394). Bispecific antigen binding molecules capable of specific binding to CD40 and Fibroblast Activation Protein (FAP) are described in WO 2018 / 185045 A1, WO 2020 / 070041 A1 or WO 2020 / 070035 A1. Bispecific FAP-CD40 antibodies for use in increasing the density of intratumoral DC- LAMP+ DCs and B cells and activating the immune system in the tumor Agonistic antibodies to CD40 expressed on APCs, in particular DCs, upregulate tumor antigen-processing and presentation and enhance the cross-priming of tumor-specific cytotoxic T-lymphocyte responses. The bispecific FAP-CD40 antibodies described herein combine a moiety capable of binding to FAP with a moiety capable of agonistic binding to CD40, wherein the activation of antigen presenting cells (APCs) through CD40 is provided by cross-linking through FAP expressed on tumor stroma cells and potentially also through FAP intermediately expressed in secondary lymphoid tissues. In contrast to bispecific antigen binding molecules capable of specific binding to CD40 and to immune checkpoint receptors on activated T cells, such as CTLA-4 or PD-1, targeting to a tumor target such as FAP enables CD40-mediated APC activation mainly in the tumor stroma and tumor-draining lymph nodes where fibroblasts express increased levels of FAP compared to other tissues. The bispecific FAP-CD40 antibodies are thus able to trigger the CD40 receptor not only effectively, but also very selectively at the desired site while overcoming the need for FcγR cross-linking thereby reducing side effects. It is shown herein that bispecific FAP-CD40 antibodies increased the density and activation of mature DC-LAMP+ DCs as well as the B cell density and organization in TLS-like foci in tumors. Dendritic cells (DCs) form a heterogenous cell population represented by many subsets. They are distinguished based on different transcription factors, surface molecules, and functions within the tumor microenvironment. The expression of DC-LAMP (CD208), characterizes mature DCs. Studies regarding melanomas reported that a high density of DC-LAMP+ cells in sentinel lymph nodes was associated with prolonged survival of patients. Positive correlations between counts of DC-LAMP+ dendritic cells and T-cell infiltration within breast cancer tissues were also reported. The presence of DC-LAMP+ cells was also associated with favorable outcomes for these patients (Dyduch et al., Int. J. Mol. Sci.2023, 24(3), 1933). It is also known that a high density of tumor-infiltrating CD8+ T cells and CD20+ B cells correlates with prolonged survival in patients with a wide variety of human cancers.The first in human (FIH) study using a bispecific FAP-CD40 antibody has been concluded in patients with advanced solid tumors. The present disclosure is based on the key outcomes of the biomarker analysis. The pharmacodynamic effects in tumor tissue were investigated in-depth in two dedicated biomarker dose expansion cohorts designed to collect tumor biopsies before and after treatment (number of patients enrolled with mandatory biopsy collection: 19 and 24 at 140 mg and 550 mg dose, respectively). Treatment with a a bispecific FAP-CD40 antibody resulted in a remarkable increase of DC-LAMP+ DC density in tumor tissue, confirming intratumoral pharmacodynamic activity and proof of mechanism, supporting the involvement of CD40 agonism in the maturation of DC subsets in the tumor microenvironment. Moreover, FAP-CD40 antibody treatment led to a notable increase in intratumoral B cell density and formation of pre-tertiary lymphoid structures (pre-TLS), co-organized in focal micro-neighbourhoods together with DC-LAMP+ DCs. The results were validated by employing two orthogonal methods: multiplexed immunofluorescence (IF) analysis and next generation RNA sequencing analysis. Together, these results provide robust evidence of significant modulation of the tumor microenvironment through FAP-targeted CD40 crosslinking. Targeting CD40 agonism to the tumor achieved a strong and sustained target engagement and tumor tissue immunomodulation, supporting further studies in combination with other anti-cancer agents in early lines of cancer treatment. Thus, provided herein is a bispecific FAP-CD40 antibody for use in treating cancer in combination with at least one anti-cancer agent, wherein the bispecific FAP-CD40 antibody increases the density of intratumoral DC-LAMP+ DCs and B cells and activates the immune system in the tumor so that it is more sensitive to treatment with the anti-cancer agent. Bulk RNA-Seq analysis of paired biopsy tissues confirmed increased expression of genes specific for B cells, activated dendritic cells (DC-LAMP+ DCs) and FAP-CD40 induced immune activation Bulk RNA sequencing analysis was used to confirm the modulation of the immune system in the tumor. Expression levels of selected signature genes measured in tumor biopsies post FAP-CD40 treatment were compared to baseline expression levels. The analysis indicated an increased expression of genes previously shown to be specific for B cells and DC-LAMP+ dendritic cells (Mädler et al., NAR Genomics and Bioinformatics 2021, 3(4), Iqab102). In addition, increased expression of genes determined in preclinical experiments to be induced in response to FAP-CD40 treatment, collectively forming a new gene signature for immune activation induced by the bispecific FAP-CD40 antibody, was determined. After treatment with bispecific FAP-CD40 antibody, RNA sequencing analysis revealed upregulation of the collective expression levels of the genes CCL22, CCL17, CCL19, HMSD,NCCRP1, UBD and CRLF2, representing genes previously determined to be specifically expressed in DC-LAMP+ dendritic cells. It also revealed upregulation of the collective expression levels of the genes CD19, MS4A1, TNFRSF13C, VPREB3, PAX5 and CR2, representing genes previously determined to be specifically expressed in B cells. Thus, the data revealed increased DC-LAMP+ and B cell specific gene expression upon FAP-CD40 treatment, in line with an increased relative proportion of these cell populations in treated versus baseline samples. In addition, the collective expression levels of the genes FAS, SEMA7A, CCL22, TRAF1, BCL2A1, IL4I1 and CFB, which we showed to be induced by FAP-CD40 treatment in preclinical experiments, were also increased in treated tumor biopsies compared to baseline levels, indicating a new signature for FAP-CD40 immune activation. Thus, provided is a bispecific FAP-CD40 antibody for use in treating cancer in combination with at least one anti-cancer agent, wherein the activation of the immune system in the tumor is determined by detecting the expression level of one or more genes selected from a group consisting of FAS, SEMA7A, CCL22, TRAF1, BCL2A1, IL4I1 and CFB from a tumor sample and comparing the expression level of these genes to a reference level, whereby an increase in the expression level is indicative for an increase of the density of intratumoral DC- LAMP+ DCs and B cells. In particular, the activation of the immune system in the tumor is determined by detecting the expression level of all genes selected from the group consisting of FAS, SEMA7A, CCL22, TRAF1, BCL2A1, IL4I1 and CFB. Provided is also an in vitro method of monitoring if a patient having a tumor is responsiveto cancer immunotherapy, the method comprising determining the activation of the immune system in a tumor tissue sample obtained from the patient having received a bispecific FAP- CD40 antibody in a first treatment regimen, wherein the activation of the immune system in the tumor is characterized by detecting the expression level of all genes selected from the group consisting of FAS, SEMA7A, CCL22, TRAF1, BCL2A1, IL4I1 and CFB and wherein the method further comprises a step of comparing the expression level of the genes to a reference level, whereby an increased expression level indicates that the tumor is more sensitive to treatment with an anti-cancer agent in a second treatment regimen.Table A (Sequences):SEQ IDName SequenceNO: 1hu CD40 VH QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQSLEWMGRVIPNAGGTSYNQKFKGRVTL TVDKSISTAYMELSRLRSDDTAVYYCAREGIYWWG QGTTVTVSS 2hu CD40 VL DIVMTQTPLSLSVTPGQPASISCRSSQSLVHSNGNTFLHWYLQKPGQSPQLLIYTVSNRFSGVPDRFSGS GSGTDFTLKISRVEAEDVGVYFCSQTTHVPWTFGG GTKVEIK 3humanized FAP (212) VH QVQLVQSGAEVKKPGASVKVSCKASGYTLTDYNMDWVRQAPGQGLEWIGDIYPNTGGTIYNQKFKGRVTM TIDTSTSTVYMELSSLRSEDTAVYYCTRFRGIHYA MDYWGQGTTVTVSS 4humanized FAP (212) VL EIVLTQSPATLSLSPGERATLSCRASESVDNYGLSFINWFQQKPGQAPRLLIYGTSNRGSGIPARFSGSG SGTDFTLTISSLEPEDFAVYFCQQSNEVPYTFGGG TKVEIK 5FAP(28H1) VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGKGLEWVSAIWASGEQYYADSVKGRFTIS RDNSKNTLYLQMNSLRAEDTAVYYCAKGWLGNFDY WGQGTLVTVSS 6FAP(28H1) VL EIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLAWYQQKPGQAPRLLIIGASTRATGIPDRFSGSGSGT DFTLTISRLEPEDFAVYYCQQGQVIPPTFGQGTKV EIK 7FAP(4B9) VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIIGSGASTYYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCAKGWFGGFN YWGQGTLVTVSS 8FAP(4B9) VL EIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKPGQAPRLLINVGSRRATGIPDRFSGSGSGT DFTLTISRLEPEDFAVYYCQQGIMLPPTFGQGTKV EIK 9VL (CD40) light chainDIVMTQTPLSLSVTPGQPASISCRSSQSLVHSNGN (charged) TFLHWYLQKPGQSPQLLIYTVSNRFSGVPDRFSGS GSGTDFTLKISRVEAEDVGVYFCSQTTHVPWTFGG GTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCL LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC 10 (P1AE1689) light chain crossQVQLVQSGAEVKKPGASVKVSCKASGYTLTDYNMD VH-Ckappa WVRQAPGQGLEWIGDIYPNTGGTIYNQKFKGRVTM TIDTSTSTVYMELSSLRSEDTAVYYCTRFRGIHYA MDYWGQGTTVTVSSASVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQWKVDNALQSGNSQESV TEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGECVH (CD40) (VHCH1 charged)QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIH Fc WVRQAPGQSLEWMGRVIPNAGGTSYNQKFKGRVTLknob_PGLALA_(P1AE1689) TVDKSISTAYMELSRLRSDDTAVYYCAREGIYWWG (VL-CH1) QGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALG CLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDE KVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPP CRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGG GGSGGGGSEIVLTQSPATLSLSPGERATLSCRASE SVDNYGLSFINWFQQKPGQAPRLLIYGTSNRGSGI PARFSGSGSGTDFTLTISSLEPEDFAVYFCQQSNE VPYTFGGGTKVEIKSSASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKKVEPKSCVH (CD40) (VHCH1 charged)QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIH Fc hole_PGLALA WVRQAPGQSLEWMGRVIPNAGGTSYNQKFKGRVTL TVDKSISTAYMELSRLRSDDTAVYYCAREGIYWWG QGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALG CLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDE KVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPP SRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGhu FAP (UniProt no. Q12884,MKTWVKIVFGVATSAVLALLVMCIVLRPSRVHNSE version 168) ENTMRALTLKDILNGTFSYKTFFPNWISGQEYLHQ SADNNIVLYNIETGQSYTILSNRTMKSVNASNYGL SPDRQFVYLESDYSKLWRYSYTATYYIYDLSNGEF VRGNELPRPIQYLCWSPVGSKLAYVYQNNIYLKQR PGDPPFQITFNGRENKIFNGIPDWVYEEEMLATKY ALWWSPNGKFLAYAEFNDTDIPVIAYSYYGDEQYP RTINIPYPKAGAKNPVVRIFIIDTTYPAYVGPQEV PVPAMIASSDYYFSWLTWVTDERVCLQWLKRVQNV SVLSICDFREDWQTWDCPKTQEHIEESRTGWAGGF FVSTPVFSYDAISYYKIFSDKDGYKHIHYIKDTVE NAIQITSGKWEAINIFRVTQDSLFYSSNEFEEYPG RRNIYRISIGSYPPSKKCVTCHLRKERCQYYTASF SDYAKYYALVCYGPGIPISTLHDGRTDQEIKILEE NKELENALKNIQLPKEEIKKLEVDEITLWYKMILP PQFDRSKKYPLLIQVYGGPCSQSVRSVFAVNWISY LASKEGMVIALVDGRGTAFQGDKLLYAVYRKLGVY EVEDQITAVRKFIEMGFIDEKRIAIWGWSYGGYVS SLALASGTGLFKCGIAVAPVSSWEYYASVYTERFM GLPTKDDNLEHYKNSTVMARAEYFRNVDYLLIHGT ADDNVHFQNSAQIAKALVNAQVDFQAMWYSDQNHG LSGLSTNHLYTHMTHFLKQCFSLSDhu FAP ectodomain+poly-lys-RPSRVHNSEENTMRALTLKDILNGTFSYKTFFPNW ISGQEYLHQSADNNIVLYNIETGQSYTILSNRTMK tag+his6-tag SVNASNYGLSPDRQFVYLESDYSKLWRYSYTATYY IYDLSNGEFVRGNELPRPIQYLCWSPVGSKLAYVY QNNIYLKQRPGDPPFQITFNGRENKIFNGIPDWVY EEEMLATKYALWWSPNGKFLAYAEFNDTDIPVIAY SYYGDEQYPRTINIPYPKAGAKNPVVRIFIIDTTY PAYVGPQEVPVPAMIASSDYYFSWLTWVTDERVCL QWLKRVQNVSVLSICDFREDWQTWDCPKTQEHIEE SRTGWAGGFFVSTPVFSYDAISYYKIFSDKDGYKH IHYIKDTVENAIQITSGKWEAINIFRVTQDSLFYS SNEFEEYPGRRNIYRISIGSYPPSKKCVTCHLRKE RCQYYTASFSDYAKYYALVCYGPGIPISTLHDGRT DQEIKILEENKELENALKNIQLPKEEIKKLEVDEI TLWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVRS VFAVNWISYLASKEGMVIALVDGRGTAFQGDKLLY AVYRKLGVYEVEDQITAVRKFIEMGFIDEKRIAIW GWSYGGYVSSLALASGTGLFKCGIAVAPVSSWEYY ASVYTERFMGLPTKDDNLEHYKNSTVMARAEYFRN VDYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQA MWYSDQNHGLSGLSTNHLYTHMTHFLKQCFSLSDG KKKKKKGHHHHHHmouse FAP (UniProt no.MKTWLKTVFGVTTLAALALVVICIVLRPSRVYKPE GNTKRALTLKDILNGTFSYKTYFPNWISEQEYLHQ P97321) SEDDNIVFYNIETRESYIILSNSTMKSVNATDYGL SPDRQFVYLESDYSKLWRYSYTATYYIYDLQNGEF VRGYELPRPIQYLCWSPVGSKLAYVYQNNIYLKQR PGDPPFQITYTGRENRIFNGIPDWVYEEEMLATKY ALWWSPDGKFLAYVEFNDSDIPIIAYSYYGDGQYP RTINIPYPKAGAKNPVVRVFIVDTTYPHHVGPMEV PVPEMIASSDYYFSWLTWVSSERVCLQWLKRVQNV SVLSICDFREDWHAWECPKNQEHVEESRTGWAGGF FVSTPAFSQDATSYYKIFSDKDGYKHIHYIKDTVE NAIQITSGKWEAIYIFRVTQDSLFYSSNEFEGYPG RRNIYRISIGNSPPSKKCVTCHLRKERCQYYTASF SYKAKYYALVCYGPGLPISTLHDGRTDQEIQVLEE NKELENSLRNIQLPKVEIKKLKDGGLTFWYKMILP PQFDRSKKYPLLIQVYGGPCSQSVKSVFAVNWITY LASKEGIVIALVDGRGTAFQGDKFLHAVYRKLGVY EVEDQLTAVRKFIEMGFIDEERIAIWGWSYGGYVS SLALASGTGLFKCGIAVAPVSSWEYYASIYSERFM GLPTKDDNLEHYKNSTVMARAEYFRNVDYLLIHGT ADDNVHFQNSAQIAKALVNAQVDFQAMWYSDQNHG ISSGRSQNHLYTHMTHFLKQCFSLSDMurine FAP ectodomain+poly-RPSRVYKPEGNTKRALTLKDILNGTFSYKTYFPNW lys-tag+his6-tag ISEQEYLHQSEDDNIVFYNIETRESYIILSNSTMK SVNATDYGLSPDRQFVYLESDYSKLWRYSYTATYY IYDLQNGEFVRGYELPRPIQYLCWSPVGSKLAYVY QNNIYLKQRPGDPPFQITYTGRENRIFNGIPDWVY EEEMLATKYALWWSPDGKFLAYVEFNDSDIPIIAY SYYGDGQYPRTINIPYPKAGAKNPVVRVFIVDTTY PHHVGPMEVPVPEMIASSDYYFSWLTWVSSERVCL QWLKRVQNVSVLSICDFREDWHAWECPKNQEHVEE SRTGWAGGFFVSTPAFSQDATSYYKIFSDKDGYKH IHYIKDTVENAIQITSGKWEAIYIFRVTQDSLFYS SNEFEGYPGRRNIYRISIGNSPPSKKCVTCHLRKE RCQYYTASFSYKAKYYALVCYGPGLPISTLHDGRT DQEIQVLEENKELENSLRNIQLPKVEIKKLKDGGL TFWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVKS VFAVNWITYLASKEGIVIALVDGRGTAFQGDKFLH AVYRKLGVYEVEDQLTAVRKFIEMGFIDEERIAIW GWSYGGYVSSLALASGTGLFKCGIAVAPVSSWEYY ASIYSERFMGLPTKDDNLEHYKNSTVMARAEYFRN VDYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQA MWYSDQNHGILSGRSQNHLYTHMTHFLKQCFSLSD GKKKKKKGHHHHHHCH1 domain ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKKVHinge region, X is S or P DKTHTCPXCPCH2 domain APELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQESTYRW SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKCH3 domain GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GIgG1, caucasian allotype ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGKIgG1, afroamerican allotype ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGKIgG2 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVE CPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAP IEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGKIgG3 ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLG DTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPP CPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVD GVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYT LPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSG QPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQG NIFSCSVMHEALHNRFTQKSLSLSPGKIgG4 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPP CPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQ FNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPS SIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNH YTQKSLSLSLGKhu CD40 (Uniprot P25942,MVRLPLQCVLWGCLLTAVHPEPPTACREKQYLINS version 200) QCCSLCQPGQKLVSDCTEFTETECLPCGESEFLDT WNRETHCHQHKYCDPNLGLRVQQKGTSETDTICTC EEGWHCTSEACESCVLHRSCSPGFGVKQIATGVSD TICEPCPVGFFSNVSSAFEKCHPWTSCETKDLVVQ QAGTNKTDVVCGPQDRLRALVVIPIIFGILFAILL VLVFIKKVAKKPTNKAPHPKQEPQEINFPDDLPGS NTAAPVQETLHGCQPVTQEDGKESRISVQERQmu CD40 (Uniprot P27512,MVSLPRLCALWGCLLTAVHLGQCVTCSDKQYLHDG version 160) QCCDLCQPGSRLTSHCTALEKTQCHPCDSGEFSAQ WNREIRCHQHRHCEPNQGLRVKKEGTAESDTVCTC KEGQHCTSKDCEACAQHTPCIPGFGVMEMATETTD TVCHPCPVGFFSNQSSLFEKCYPWTSCEDKNLEVL QKGTSQTNVICGLKSRMRALLVIPVVMGILITIFG VFLYIKKVVKKPKDNEILPPAARRQDPQEMEDYPG HNTAAPVQETLHGCQPVTQEDGKESRISVQERQVT DSIALRPLV(FAP 28H1) light chain crossEVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMS VH-Ckappa (P1AE2302) WVRQAPGKGLEWVSAIWASGEQYYADSVKGRFTIS RDNSKNTLYLQMNSLRAEDTAVYYCAKGWLGNFDY WGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTAS VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGECVH (CD40) (VHCH1 charged)QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIH Fc knob_PGLALA_(FAP WVRQAPGQSLEWMGRVIPNAGGTSYNQKFKGRVTL 28H1) (VL-CH1) (P1AE2302) TVDKSISTAYMELSRLRSDDTAVYYCAREGIYWWG QGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALG CLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDE KVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPP CRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGG GGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQ SVSRSYLAWYQQKPGQAPRLLIIGASTRATGIPDR FSGSGSGTDFTLTISRLEPEDFAVYYCQQGQVIPP TFGQGTKVEIKSSASTKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKKVEPKSCVL (muCD40) light chainDTVLTQSPALAVSPGERVTISCRASDSVSTLMHWY (charged) (P1AE5394) QQKPGQQPKLLIYLASHLESGVPARFSGSGSGTDF TLTIDPVEADDTATYYCQQSWNDPWTFGGGTKLEL KRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYP REAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC(FAP 28H1) light chain crossEVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMS VH-Ckappa (P1AE5394) WVRQAPGKGLEWVSAIWASGEQYYADSVKGRFTIS RDNSKNTLYLQMNSLRAEDTAVYYCAKGWLGNFDY WGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTAS VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGECVH (CD40) (VHCH1 charged)EVQLVESDGGLVQPGRSLKLPCAASGFTFSDYYMA Fc WVRQAPTKGLEWVASISYDGSSTYYRDSVKGRFTIknob_PGLALA_(P1AE1689) SRDNAKSTLYLQMDSLRSEDTATYYCGRHSSYFDY (VL-CH1) (P1AE5394) WGQGVMVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTL PPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGS GGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRA SQSVSRSYLAWYQQKPGQAPRLLIIGASTRATGIP DRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGQVI PPTFGQGTKVEIKSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSC33 VH (CD40) (VHCH1 charged)EVQLVESDGGLVQPGRSLKLPCAASGFTFSDYYMA Fc hole_PGLALA (P1AE5394) WVRQAPTKGLEWVASISYDGSSTYYRDSVKGRFTI SRDNAKSTLYLQMDSLRSEDTATYYCGRHSSYFDY WGQGVMVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTL PPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPG The present invention is further described by reference to the following non-limited figures and examples.Examples Example 1 First in human (FIH) study of FAP-CD40 in patients with advanced solid tumors First-in-human study WP42627 evaluated the safety, pharmacokinetics (PK), pharmacodynamics (PD), and anti-tumor activity of the FAP-CD40 bispecific antibody RO7300490, a bispecific, fibroblast activation protein alpha (FAP)-targeted CD40 (FAP-CD40) agonistic antibody, as single agent (Part 1) in adult patients with locally advanced and / or metastatic solid tumors. Study WP42627 was conducted in accordance with ICH-GCP and the Declaration of Helsinki, and was approved by the Institutional Review Boards and / or local Ethics Committees of the participating centers. All participants signed a written informed consent before enrollment. FAP-targeted CD40 agonistic antibody RO7300490 was administered on day 1 of every 2-weeks cycle via intravenous (IV) infusion and limited to 24 months or until progressive disease, unacceptable toxicities, or withdrawal of consent. Six dose level cohorts with ascending RO7300490 doses of 16 mg up to 1100 mg were assessed. The enrollment of the first 3 participants was staggered to mitigate the risk of acute toxicity. At least 3 participants were enrolled per dose level and were monitored for DLT toxicity until Cycle (C)2 Day (D)7, prior decision to dose escalate to the next cohort was made jointly between the Sponsor and the Investigators. Intrapatient dose escalation to the next safest dose was allowed after the second scheduled tumor assessment was performed. Additional participants were subsequently enrolled in 2 biomarker dose extension cohorts, at 140 mg or 550 mg RO7300490 respectively, in order to collect paired baseline and on-treatment (at C3D3) biopsies for PD analysis. The study enrolled adult participants > / = 18 years old, diagnosed with advanced and / or metastatic solid tumors who progressed on previous cancer therapy / ies or were not amenable to standard therapy, following informed consent. Tumor types known to express FAP, including non small cell lung cancer (NSCLC) small cell lung cancer (SCLC) triple negative breast cancer (TNBC), cutaneous melanoma, urothelial cancer, mesothelioma, hepatocellular carcinoma (HCC), head and neck squamous cell carcinoma (HNSCC), esophageal squamous cell carcinoma (ESCC), and cervical squamous cell carcinoma, were preferred. Further solid tumor types were accepted on a case by case presentation. Patients had to have an Eastern Cooperative Oncology Group Performance Status (ECOG) of 0-1; adequate cardiovascular (provide cutoffs), bone marrow (provide), renal (provide), hepatic (provide), coagulation (provide) functions, and measurable disease per Response Evaluation Criteria in Solid Tumors (RECIST) V.1.1. Patients who were enrolled to the biomarker extension cohorts had to have lesions that could be safely biopsied, and specifically consent to the biopsy procedures.Patients were excluded from the study if they had symptomatic or unstable CNS tumors or metastases; untreated spinal cord compression; significant cardiovascular / cerebrovascular disease within 6 months prior enrollment; known hereditary or acquired coagulopathies; history of idiopathic pulmonary fibrosis; history or evidence of pneumonitis / interstitial lung disease; active or history of autoimmune disease at the exception of medically managed autoimmune mediated hypothyroidism, or hypopituitary or adrenal insufficiency, or type 1 diabetes mellitus; prior allogeneic transplant; chronic or active human immunodeficiency virus (HIV), or hepatitis B virus (HBV), or hepatitis C virus (HCV) infection; history of severe allergic or anaphylactic reactions to mAb or recombinant antibody-related fusion proteins or components of their formulation; systemic immunosuppressive medications within 14 days prior the first dose with the exception of acute and / or low-dose systemic immunosuppressive medications under certain conditions. Treatment with systemic immune-modulating agents and other anticancer agents had to wash off before the first dose of study treatment. Example 2 RO7300490 (FAP-CD40) increased the density and activation of mature DC-LAMP+ DCs as well as the B cell density and organization in TLS-like foci in tumors The pharmacodynamic effects of RO7300490 were investigated in two dedicated biomarker dose expansion cohorts (140 mg and 550 mg dose cohort, respectively) designed to collect tumor biopsies before and after treatment. All tissue samples were collected, processed and paraffin-embedded according to the standardized and approved histopathology protocols. Formalin-fixed paraffin-embedded (FFPE) tumor tissue blocks were sectioned consecutively either at three microns (for immunofluorescence assay) or at four microns (for hematoxylin and eosin stain (H&E)). Tumor biopsies were collected before treatment and 48 hours after the 3rd treatment cycle, preferentially from the same biopsy location. Only samples passing quality control for sufficient tumor content without necrotic areas and excluding lymph node tissue contamination were included in the analysis. A validated custom developed 8-plex Immunofluorescence (IF) panel (LAMP3 / CLEC9a / CLEC10a / CD20 / CD70 / CD68+163 / CD86 / CD279) was performed applying Ultivue technology, followed by an additional H&E staining on the same slide. FFPE sections were stained with a locked staining protocol on the BOND RX staining platform. The following antibodies were used: CD20 (Thermo #MA5-13141), CD70 (Novus #MAB2738), CD68 / CD163 (Biocare / Abcam #CM033CF / ab182422), CD86 (CST #91882), CD279 (Abcam #ab228415), Clec9a (Abcam #ab223188), Clec10a (OriGene #TA810180), LAMP3 (Fisher Scientific #DDX0191P100). Whole slide images were digitized at 20×magnification. Round 1 and 2 images were co-registered and stacked with Ultivue’s UltiStacker software. Upon annotation, defined cell types were quantified by a validated automatic image analysis scoring algorithm using Visiopharm Software and QCed by a medical pathologist. Two samples from the 140 mg cohort were excluded due to residual lymph node tissue present in the biopsy. For each cell type and feature (cell density, density of CD86+ - coexpressing cells), samples were excluded if only one timepoint (either baseline or on treatment) was available. As the data do not follow a normal distribution and show some extreme values that could not be classified as outliers due to lack of abnormalities in the IF images, the Wilcoxon Signed Rank Test was used to determine the statistical significance of baseline vs. on- treatment comparisons. Due to the high variability inherent to IHC data and the different potential biological considerations for multiple testing corrections, raw p-values are reported. Corrected p-values were also computed for each feature using the FDR method, after checking for potential high correlations (correlation coefficient ≥ 0.8) between the 5 different cell types. The markers used for IF analysis were optimized for specific identification of DC-LAMP+ mature DCs, and CLEC9A+ classical type 1 DCs (cDC1). IF method development and validation confirmed that DC-LAMP (=LAMP3) and CLEC9A are specific, non-overlapping markers suitable for DC subset identification. In general, pharmacodynamic effects observed were similar for both doses tested. Due to inherently high variability of the cells of interest, particularly DCs, data were analyzed by pooling both dose cohorts. In this study we observed that RO7300490 (FAP-CD40) treatment resulted in a remarkable increase of DC-LAMP+ DC density in tumor tissue (DC-LAMP+ DC density log2 fold-change = 2.56, p=0.001 (Fig.1A). The increased density of DC-LAMP+ DCs coincided with a reduction of CLEC9a+ classical type 1 DCs (cDC1) (cDC1 density log2 fold-change = 1.23, p=0.02 (Fig. 1B). RO7300490 treatment also increased the activation status of DC-LAMP+ DCs co- expressing the activation marker CD86. The treatment-induced increase in the density of the DC- LAMP+ DCs was clearly visible on IF images due to their scarcity in baseline biopsy samples, as shown in representative images from a patient with Breast Cancer (pt.1008) and a patient with Mesothelioma (pt.1000) (Fig.1C). In addition, Figures 2A to 2C show that FAP-CD40 increased the B cell density andorganization in Tertiary Lymphoid Structures (TLS)-like foci in tumors. FAP-CD40 treatment led to a notable increase in intratumour B cell density (B cell density log2 fold-change = 1.84, p=0.058) (Fig.2A). IF images of two exemplary patients show increased numbers of B cell foci and enlargement of B cell foci after treatment (Fig.2B). Further analysis revealed that frequently DC-LAMP+ DCs were co-localized in scatters or in foci together with B cells, coinciding in small lymphocytic aggregates reminiscent of early TLS (Fig.2C).Taken together, the data described in this example show direct targeting of CD40 expressing cell populations with RO7300490 (FAP-CD40) in tumor tissue, successfully modulating the tumor microenvironment. FAP-CD40 increased the density and activation of mature DC-LAMP+ DC in tumors, supporting the involvement of CD40 agonism in the maturation of DC subsets in the tumor microenvironment. The data also show that FAP-CD40 treatment increased the B cell density in tumors and led to the appearance of large B cell foci colocalized with DC-LAMP+ DCs, forming structures reminiscent of nascent TLS. Example 3 Bulk RNA-Seq analysis of paired biopsy tissues confirms increased expression of genes specific for B cells, activated dendritic cells (DC-LAMP+ DCs) and FAP-CD40 induced immune activation The pharmacodynamic effects of RO7300490 were further investigated in the same two dedicated biomarker dose expansion cohorts as described above, using RNA-seq. All tissue samples were collected, processed and analyzed according to standard protocols and analytical pipelines, as described below (Example 3.2.1). Tumor biopsies were collected before treatment and 48 hours after the 3rd treatment cycle, preferentially from the same biopsy location. Only samples passing quality control for sufficient tumor content without necrotic areas and excluding lymph node tissue contamination were included in the analysis. Across patients, and in the individual dose-specific cohorts, respectively, we observed an induction of expression level of genes specific for B cells, DC-LAMP+ dendritic cells, as well as FAP-CD40-mediated immune activation (Figure 9). B cell and DC-LAMP+ dendritic cell specific genes were previously derived based on single-cell RNA-seq data and publicly available (Mädler et al., NAR Genomics and Bioinformatics 2021, Vol.3(4), https: / / doi.org / 10.1093 / nargab / lqab102). Genes specific for FAP-CD40-mediated immune activation genes were derived as described below (Figures 6 to 8) prior to and independent of the clinical study, using a set of preclinical experiments, which included two mouse in vivo studies and multiple human tumor explant studies (Figures 3 to 5). Observed RNA expression patterns were thus in line with an increased relative fraction of B cells, DC-LAMP+ dendritic cells and overall FAP-CD40-mediated immune activation. 3.1. Gene expression analysis of samples isolated from mouse tumor studies and human tumor explants 3.1.1. Gene analysis in MC38-FAP tumors upon treatment with an anti-FAP / anti- CD40 mouse surrogate molecule (P1AE2302-039) by Nanostring MC38 cells (murine colon adenocarcinoma tumor) were obtained from the City of Hope (Duarte, California, US) and engineered in house to express the mouse fibroblast activationprotein alpha (MC38-muFAP). Following an in-vivo passage and after further expansion, the MC38-muFAP-invipa tumor cell line was deposited in the Glycart internal cell bank. Cells were cultured in Dulbecco's Modified Eagle Medium containing 10% fetal calf serum (FCS; PAA Laboratories, Austria), 1 mM pyruvate, 1× non-essential amino acids solution, and 6 μg / mL puromycin at 37°C in a water-saturated atmosphere at 5% CO2. HumanCD40Tg homozygous female mice (purchased from Charles Rivers, France, and originally obtained from Taconic), were maintained under specific-pathogen-free conditions at 12:12-hour light:dark cycles according to committed guidelines (GV-Solas, Felasa, TierschG). The experimental study protocol was reviewed and approved by the local government (ZH225- 17). After arrival, animals were maintained for 1 week to acclimatize to the new environment and for observation. They were afterwards implanted subcutaneously with a transponder on the right side of their back for identification and maintained for an additional week for recovery. Health monitoring was carried out on a regular basis. Animals were controlled daily for clinical symptoms and detection of adverse effects. Termination criteria for animals were specified in the corresponding animal license (ZH225-17). Gene expression was analyzed upon treatment with an anti-FAP / anti-CD40 bispecific mouse surrogate molecule (P1AE2302-039) by NanoString analysis.2x106MC38-muFAP- invipa tumor cells were injected at in vitro passage 11 at a viability of 96.5% in a total volume of 100-μL in a 1:1 mix with RPMI and Matrigel (734-0269, Corning, VWR International GmbH), subcutaneously into huCD40 tg mice on study day -24 (Fig.3). Tumor growth was monitored by bi-weekly caliper measurements. At day twenty-four, when the mean tumor volume reached 260 mm3, the mice were injected intraperitoneally in the vehicle group with Histidine buffer or with 13.3 mg / kg P1AE2302 in the treatment group. One day after injection, the tumor was collected from three mice per group and snap frozen in liquid nitrogen. To isolate total RNA, fresh-frozen tumor samples were homogenized in the RNA lysis buffer, and RNA was extracted using the RNEasy Mini kit (74106, Qiagen). Gene expression was quantified using the NanoString nCounter® platform, by hybridizing 200 ng of total RNA to the nCounter® Mouse Immunology Panel, comprising 561 immunology-related mouse genes (115000052, NanoString Technologies) at 65°C overnight. RNA transcripts were immobilized and counted using the NanoString nCounter® Digital Analyzer. The LIMMAT platform, implemented in-house, was used for statistical analysis of the gene-expression data. To test for significant differences in group means by multiple comparisons, standard ANOVA was used. 3.1.2. Gene expression analysis in KPC-4662-huCEA tumors and lymph nodes upon treatment with an anti-FAP / anti-CD40 bispecific mouse surrogate molecule (P1AE5394-122) by scRNA- seq analysis KPC-4662 cells (murine pancreatic tumor) were obtained from the University of Pennsylvania and engineered in house to express the human carcinoembryonic antigen (KPC-4662-huCEA). Cells were cultured in DMEM + 10% FCS (PAA Laboratories, Austria) + 500 ug / mL Hygromicin at 37 °C in a water-saturated atmosphere at 5 % CO2. C57Bl / 6-huCEA tg female mice (purchased from Charles Rivers, France), were maintained under specific-pathogen-free condition with daily cycles of 12 h light / 12 h darkness according to committed guidelines (GV-Solas; Felasa; TierschG). The experimental study protocol was reviewed and approved by the local government (ZH225-17). After arrival, animals were maintained for one week to get accustomed to the new environment and for observation. They were afterwards implanted with a transponder subcutaneously on the right side of the back for identification and maintained one more week for recovery. Continuous health monitoring was carried out on a regular basis. Animals were controlled daily for clinical symptoms and detection of adverse effects. Termination criteria for animals were specified in the corresponding animal license (ZH225-17). Gene expression was analyzed upon treatment with an anti-FAP / anti-CD40 bispecific mouse surrogate molecule (P1AE5394-122) by single-cell RNA-seq.3x105 KPC-4662-huCEA cells were injected at in vitro passage 6 at a viability of 97% in a total volume of 100-μL in a 1:1 mix with RPMI and Matrigel (734-0269, Corning, VWR International GmbH), subcutaneously into C57Bl / 6-huCEA tg mice on study day 0 (Fig.4). Tumor growth was monitored by bi- weekly caliper measurements. At day twenty-one, when the mean tumor volume reached an average size of 200 mm3, the mice were injected intraperitoneally in the vehicle group with Histidine buffer or with 13.3 mg / kg P1AE5394-122 in the treatment group. Twenty-four and forty-eight hours after treatment, 5 mice per group and per timepoint were sacrificed and the tumor as well as inguinal draining lymph nodes were collected in PBS for subsequent cell sorting and sc RNA-seq. In a first step, single cell suspensions were obtained by digesting the organs for 10 min at 37 °C using a mix containing 0.05 mg / ml DNAse (Roche, 11284932001) and 1 mg / ml Collagenase D (Roche, 11088858001). Red blood cell lysis (BD 555899) was performed when required. In order to pre-enrich the tumor samples a Percoll (GE Healthcare density 1.130 g / mL, 17-0891-02) density gradient centrifugation was done before sorting. In a 15 mL tube, the cell pellet was resuspended in 10 mL of 40% Percoll and slowly layered on top of 70 % Percoll. The tubes were centrifuged for 20 minutes at 2500 rpm with acceleration and deceleration set to 0. Then, the leukocyte ring was collected, transferred into a 15 mL tube and washed with medium. Cells were then frozen in 10 Mio / ml cells per ml in Ibidi freezing medium classic (Ibidi, Cat. No.80022). On the day of the sorting, the single cell suspensions of tumor and lymph nodes were thawed, transferred into 96-well round-bottom plates, washed with PBS and incubated with 50 µl of Fc receptor blocking mouse IgG Isotype Control (3 µg / ml, ThermoFisher Scientific, Cat. No.10400C) in PBS. After 15 minutes incubation at 4°C, cells were washed with PBS and 50 µl of a mixture of fluorescently labeled antibodies in PBS was added.Cells were stained with the following markers: MHC II Pacific Blue (eBioscience #48- 5321-82), Live / dead NearlR (ThermoFisher Scientific #L34976), CD11c APC (BD Bioscience #550261), B220 FITC (Biolegend #103206), F480 PeCy5 (Biolegend #123112), NK1.1 PE (eBioscience #12-5941-82), CD3 PE CF594 (BD Bioscience #562286), and CD45 AF700 (eBioscience #56-0451-82). Cells were then sorted using the 100µm nozzle on the BDFACS Aria sorter. To distinguish major cell populations, (i) myeloid cells were determined as CD45+ CD3- NK1.1- cells (which were composed of CD45+ CD19+ B220+ CD11c- B cells, CD45+ F480- MHCII+ CD11c+ DCs and CD45+ F480+ macrophages / monocytes), (ii) T cells as CD45+ CD3+ cells, and (iii) NK cells as NK1.1+ cells. T cells and NK cells were sorted in one vial, and the myeloid cells were sorted in a separate tube. For all samples, sc RNA-seq was performed using Chromium Next GEM Automated Single Cell 3’ Library and Gel Bead Kit v3.1 (10x Genomics, Pleasanton, CA, USA) following the manufacturer’s protocol. A maximum of 20000 (CD3- NK1.1- CD45+) myeloid cells were used for further scRNA- seq analysis. For the samples where less than 20000 myeloid cells were available, the maximum of sorted cells was pooled with the T and NK cell fraction and further processed for scRNA-seq analysis. Cells diluted in PBS plus 1% BSA (determined by Cellometer Auto 2000 Cell Viability Counter (Nexcelom Bioscience, Lawrence, MA USA)), were loaded into the 10x Chromium Controller and library preparation was performed according to the manufacturer’s indications (Chromium Next GEM Automated Single Cell 3’ Library and Gel Bead Kit v3.1, Chromium Next GEM Automated Chip G Single Cell Kit, Single Index Kit T Set A (all 10x Genomics), SPRIselect Reagent Kit (Beckman-Coulter)). The quality and concentration of both cDNA and libraries were assessed using an Agilent BioAnalyzer with High Sensitivity kit (both Agilent, Santa Clara, CA USA) and Qubit Fluorometer with dsDNA HS assay kit (both Thermo Fisher Scientific, Waltham, MA USA) according to the manufacturer’s recommendation. The libraries were pooled equimolarly and clustered using an Illumina cBOT instrument (HiSeq 3000 / 4000 PE Cluster Kit) and sequenced on an Illumina HiSeq 4000 (HiSeq 3000 / 4000 SBS Kit, 150 cycles) with a depth of ~50,000 reads / cell and the recommended read configuration (R1=28, I7=8 and R2=91). The output files were converted to FASTQ files using the Cell Ranger pipeline. Further processing was performed as described below (see Example 3.2.1).3.1.3. Gene analysis in cell sorted from human tumorGene expression was analyzed in cell populations sorted from human tumor explant samples upon treatment with a FAP-CD40 human surrogate molecule (P1AE2423-020) or left untreated. All patients signed a written informed consent before sample collection. Ethical approvals were given as follows: four tumor samples were collected at the University Hospital Zurich or Hirslanden Hospital, Zurich, and approved by the local government ethical commission Kanton Zurich. One sample was obtained commercially through Indivumed Services. None of the experiments were blinded. For baseline characterization, patient samples were mechanically dissociated and digested using accutase (PAA Laboratories, #L11-007 or Sigma-Aldrich #A6964), collagenase IV (Worthington, #LS004188), hyaluronidase (Sigma-Aldrich, #H6254) and DNAse type IV (Sigma-Aldrich, #D5025). Then, tumors were stained for flow cytometry with the following markers: CD45 BV510 (Biolegend #304036), CD3 PE-Cy5 (BD Biosciences #555341), CD11c BV421 (Intracellular staining, Biolegend #301628), CD56 FITC (Biolegend #318304), CD14 PerCP-Cy5.5 (Biolegend #325622), HLA-DR BV786 (BD Biosciences #564040), CD19 AF700 (BD Biosciences #562652), CD16 PE-Cy7 (Biolegend #561132(, CD40 BV650 (Biolegend #334324), FAP AF647 (Tapir ID P1AD4900-004) and viability (LIVE / DEAD™ Fixable Near IR (780) Viability Kit, Invitrogen). Tumor digest was fixed and permeabilized according to manufacturers recommendation (eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set, #00-5523-00). Tumor tissue pieces (1-2 mm3) were placed in a bioreactor (Muraro et al., Oncoimmunology 2017, 6(7), e1331798), supplied with with culture medium [DMEM / F12 (Gibco Life Technologies #31331) supplemented with 1 mM sodium pyruvate, 1% MEM nonessential amino acids, 2 mM L-glutamine, 5% human serum, 10mM HEPES] and treated with 3nM (or 6.7 nM) FAP-CD40 (P1AE2423-020) between 18h and 72h or left untreated. Some samples were additionally supplemented with either 2 ng / ml GM-CSF (Peprotech, #300-03), or treated with or without 100 ng / ml Fltr3L (Peprotech, #300-19) and 20 ng / ml IL-3 (Peprotech, #200-003) (Fig.5). After cultivation the tumor pieces were enzymatically digested as described above and stained for FACS sorting using CD45 BV510 (Biolegend #304036), CD3 PE-Cy5 (BD Biosciences #555341), CD56 FITC (Biolegend #318304) and viability (LIVE / DEAD™ Fixable Near IR (780) Viability Kit, Invitrogen). For all samples (as described in Fig. 5), sc RNA-seq was performed using ChromiumSingle Cell 3’ GEM, Library & Gel Bead Kit v3 (10x Genomics, Pleasanton, CA, USA)following the manufacturer’s protocol. Briefly, up to 16000 CD45+ immune cells or CD45+ immune cells depleted for T and NK cells per sample, diluted in PBS plus 1% BSA (determined by Cellometer Auto 2000 Cell Viability Counter (Nexcelom Bioscience, Lawrence, MA USA)), were loaded into the 10x Chromium Controller and library preparation was performed according to the manufacturer’s indications (Chromium Single Cell 3’ Library & Gel Bead Kit v3, i7 Multiplex Kit, Single Cell B Chip (all 10x Genomics), SPRIselect Reagent Kit (Beckman- Coulter)). The quality and concentration of both cDNA and libraries were assessed using an Agilent BioAnalyzer with High Sensitivity kit (both Agilent, Santa Clara, CA USA) and Qubit Fluorometer with dsDNA HS assay kit (both Thermo Fisher Scientific, Waltham, MA USA) according to the manufacturer’s recommendation. The libraries were pooled equimolarly and clustered using an Illumina cBOT instrument (HiSeq 3000 / 4000 PE Cluster Kit) and sequenced on an Illumina HiSeq 4000 (HiSeq 3000 / 4000 SBS Kit, 150 cycles) with a depth of ~50,000 reads / cell and the recommended read configuration (R1=28, I7=8 and R2=91). The output files were converted to FASTQ files using the CellRanger pipeline. Further processing was performed as described below (see Example 3.2.1).3.1.4 Gene expression analysis in tumor biopsies at baseline and upon FAP-CD40 (RO7300490 ) treatment All tissue samples were collected, processed and analyzed according to standard protocols and analytical pipelines, as described below. Tumor biopsies were collected before treatment and 48 hours after the 3rd treatment cycle, preferentially from the same biopsy location. Only samples passing quality control for sufficient tumor content without necrotic areas and excluding lymph node tissue contamination were included in the analysis. Total RNA was isolated from microdissected formalin-fixed paraffin-embedded tumor tissue sections using the Qiagen AllPrep DNA / RNA FFPE kit according to the manufacturer’s instructions. RNA was subsequently subjected to library preparation with the Illumina TruSeq RNA Exome Kit which is a hybridization-based assay to enrich coding RNAs from total RNA sequencing libraries. Libraries were sequenced on a NovaSeq 6000 instrument (Illumina) at a targeted read depth of 25 M per sample. Base calling was performed with BCL to FASTQ file converter bcl2fastq2 version 2.20.0 (Illumina). 3.2 RNA-Seq data analysis of preclinical experiments and clinical tumor biopsies 3.2.1. Human and mouse scRNA-seq data analysis Fastq files were aligned to the human (GRCh38) or mouse (mm10) transcriptome, respectively using CellRanger count 3.1.0 (human tumor explant data), v4.0.2 (mouse in vivo data) with the parameters --expect-cells = 6000’. All cells showing >200 counts were further merged across all samples and processed with scanpy (large-scale single-cell gene expressiondata analysis, Wolf et al., Genome Biology 2018, 19, 15) and the besca standard worklfow (single-cell transcriptomics analysis toolkit to accelerate translational research, NAR Genom. Bioinform.2021, 3(4), Iqab102). Filtering was performed with the parameters (a) min_genes =500, min_cells = 30, min_counts = 1000, n_genes = 7000, percent_mito = 0.20,max_counts = 80000 for the mouse in vivo data (exp1); (b) min_genes =800, min_cells =10,min_counts = 1500, n_genes = 5000, percent_mito = 0.15, max_counts =30000 for the tumorexplant data on which we focused on the B cell analysis (Pat5, exp2); min_genes =500,min_cells =10, min_counts = 1000, n_genes = 5000, percent_mito = 0.15, max_counts =25000 for the human tumor explant data on which we focused on the myeloid cell analysis (Pat1-4,exp3, see Example 3.1.3).In brief, RNA counts were normalized per 10,000, the top most highly variable genes were selected, total gene and mitochondrial reads were regressed out, PCA was performed and the first 50 principal components were used for nearest-neighbor calculations and Leiden clustering, as well as for UMAP-based visualization. Annotation was performed using besca’s sig-annot module, attributing clusters to distinct cell types based on specific enrichment of marker gene expression using the signatures provided with the package. Differential expression analysis wasperformed per cell type at distinct annotation levels. In the mouse in vivo experiment (exp1, seeExample 3.1.2) differential expression analysis on pseudobulk expression profiles using edgeR 3.32.1 in R 4.0.1 and the model ~treatment * relative_timepoint; samples originating from lymph node and tumor were analyzed separately. Differential expression was performed per cell subset (dendritic cells, macrophages, B cells) or main cell type (all available immune cell populations, termed “immune cells”). Human samples were analyzed separately (transcriptionally selected Blymphocytes from a single donor [Pat 5] at three distinct time-points, exp2; transcriptionallyselected myeloids from 4 distinct donors [Pat 1-4], (exp3, see Example 3.1.3), and fold-changeswere calculated per condition (either time-point or donor) and cell (sub)type (B cells, cDCs, or myeloid cells); significance of differential expression was assessed using the non-parametric Wilcoxon rank-sum test as implemented in scanpy (method=”wilcoxon”, FDR 0.05). 3.2.2. Selection of the FAP-CD40 activationBased on differential expression in FAP-CD40 treated versus control samples in thedistinct preclinical scRNA-seq datasets (Figures 4 and 5, Examples 3.1.2 and 3.1.3), thepreclinical Nanostring immunology-related mouse gene analysis (Figure 3, Example 3.1.1) and publicly available CD40 perturbation data in a human cell line (Basso et al., Blood 2004, 104(13), 4088-4096), a set of genes was selected for an initial inspection of fold-changes upon FAP-CD40 treatment. Putative B cell activation markers were selected as significantly differentially expressed (DE) at any timepoint (24h or 48h) across cells identified based on their gene expression pattern as B cells as well as across all analyzed immune cells of the experiment(in LN or Tumor, exp1; fold-change >=1.5 and FDR 0.05) and upregulated in at least one humantumor explant experiment across transcriptionally identified B cells (any timepoint, exp2, FDR0.05, FC>=1.5) or present in the list of CD40-specific genes provided by Basso et al. (putative Bcell activation markers: Bcl2a1b, Map3k8, Nfkbie, Nfkbid, Il4i1, St14). Putative cDC activationmarkers were selected as DE at any timepoint (24h or 48) across transcriptionally identified cDCas well as across all available immune cells (in LN or Tumor, exp1, fold-change >=1.5 and FDR0.05) and upregulated in the Nanostring expresiment (FDR 0.05) or present in the Basso et al.list (putative cDC activation markers: Ccl22, Cd40, Traf1, Ehd1, Nfkbie, Srgn, Cfb, Ifi204, Il1a).Putative general activation markers were selected as DE at any timepoint (24h or 48h) across transcriptionally identified cDCs / macrophages as well as across all immune cells (LN or Tumor,exp1, fold-change >=1.5 and FDR 0.05) and one of the tumor explant experiments (B cells,cDCs or myeloids, exp3, fold-change >=1.5 and FDR 0.05) or upregulated in the Nanostring data(putative general activation markers: Ccl22, Cd40, Traf1, Ehd1, Nfkbie, Fas, Sema7a, Srgn,Hspa1a, Ifi204). All these genes and additional set of genes related to CD40 signaling documented in literature (Cd83, Cd86, Cd80, Cd69, Cd79, Cxcl9, Cxcl10) were inspected for cell type specific expression patterns in the mouse preclinical data (Fig.6). Subsequently, two genes with highly abundant and unspecific expression (Srgn, Cd69) were removed from the list. We display the fold-change of these genes across tumor-infiltrated immune and dendritic cellsderived from FAP-CD40 treated versus control mice (exp1) in Fig. 7. Next, we retained thegenes that showed a fold-change of >=2 in one of the human tumor experiments (exp2 or exp3)(Fig. 8), resulting in a final gene list of FAS, SEMA7A, CCL22, TRAF1, BCL2A1, IL4I1, CFB,which we refer to as “FAP-CD40 immune activation signature” or “FAP-CD40_up_i”, in short. 3.2.3. Patient bulk RNA-seq The pharmacodynamic effects of RO7300490 were further investigated in the same two dedicated biomarker dose expansion cohorts as described above, using RNA-seq. All tissue samples were collected, processed and analyzed according to standard protocols and analytical pipelines, as described below. Tumor biopsies were collected before treatment and 48 hours after the 3rd treatment cycle, preferentially from the same biopsy location. Only samples passing quality control for sufficient tumor content without necrotic areas and excluding lymph node tissue contamination were included in the analysis. Total RNA was isolated from microdissected formalin-fixed paraffin-embedded tumor tissue sections using the Qiagen AllPrep DNA / RNA FFPE kit according to the manufacturer’s instructions. RNA was subsequently subjected to library preparation with the Illumina TruSeq RNA Exome Kit which is a hybridization-based assay to enrich coding RNAs from total RNA sequencing libraries. Libraries were sequenced on a NovaSeq 6000 instrument (Illumina) at a targeted read depth of 25 M per sample. Base calling was performed with BCL to FASTQ file converter bcl2fastq2 version 2.20.0 (Illumina). FASTQ files were quality checked with FastQC version 0.11.9 (Andrews S. (2010). FastQC: a quality control tool for high throughput sequencedata. Available online at: http: / / www.bioinformatics.babraham.ac.uk / projects / fastqc). RNA-seq paired-end reads were mapped onto the human genome (hg38) with the read aligner STAR version 2.7.3a using default mapping parameters (see Dobin et al., Bioinformatics 2013, 29(1), 15-21). Aligned reads were quality checked with MultiQC version 1.9 (see Ewels et al., Bioinformatics 2016, 32(19), 3047-3048). Numbers of mapped reads for all RefSeq and / or Ensembl transcript variants of a gene were combined into a single value (count) by featureCounts version 2.0.1 (see Liao et al., Bioinformatics 2014, 30(7), 923-930). Counts were computed assuming a reverse-stranded library and normalized as transcripts per million (TPM). All samples passed quality control and were included in the analysis, with the exception of those coming from three patients that did not pass histology criteria (as they showed either cytology-like morphology on baseline or residual lymph node tissue). Genes expressed at >1 counts per million (CPM) in at least 8 samples were included and further processed in R version 4.2.0 (R core team

[2022] ) and the packages limma 3.54.2 (see Ritchie et al., Nucleic Acids Res. 2015, 43(7), e47), and edgeR 3.40.2 (Robinson et al., Bioinformatics 2010, 26(1), 139-140). Differential gene expression analysis was performed using the voom-limma method and the model ~ 0 + Patient + Visit (Law et al., Genome Biology 2014, 15(2), R29. https: / / doi.org / 10.1186 / gb-2014-15-2-r29) and signature enrichment analysis performed using CAMERA (Wu et al., Nucleic Acids Res.2012, 40(17), e133). Three signatures were used (Fig. 9): the FAP-CD40 activation signature, derived as described above (FAP-CD40-up_i: FAS, SEMA7A, CCL22, TRAF1, BCL2A1, IL4I1, CFB), and two cell-type specific signatures previously derived based on single-cell RNA-seq data (see Mädler et al., NAR Genomics and Bioinformatics 2021, Vol.3(4), https: / / doi.org / 10.1093 / nargab / lqab102) (cDC_CCR7_sc / activated dendritic cell signature: CCL22, CCL17, CCL19, HMSD, NCCRP1, UBD, CRLF2; a Bcell_sc / B cell signature: CD19, MS4A1, TNFRSF13C, VPREB3, PAX5, CR2). Summary of Results Across patients, and in the individual dose-specific cohorts, respectively, we observed an induction of expression level of genes specific for B cells, DC-LAMP+ dendritic cells, as well as FAP-CD40 mediated immune activation (Figure 9). FAP-CD40 activation genes were derived as described herein before (Figures 6 to 8) prior to the clinical study from a set of preclinicalexperiments, which included two mouse in vivo studies and multiple human tumor explantstudies (see Examples 3.1.1 to 3.1.3, Figures 3 to 5). B and DC-LAMP+ dendritic cell specificgenes were previously derived based on sc RNA-seq data (Mädler et al., NAR Genomics and Bioinformatics 2021, Vol.3(4), https: / / doi.org / 10.1093 / nargab / lqab102). Observed RNA expression patterns were thus in line with an increased relative fraction of B cells, activated dendritic cells and overall FAP-CD40-mediated activation.Taken together, the FAP-CD40 induced increase in density and activation status of DC- LAMP+ DCs in tumor tissue using a validated multiplex immunofluorescence method was a key intra-tumoral pharmacodynamic outcome of the first-in-human study of FAP-CD40 in patients with advanced solid tumors. Another notable pharmacodynamic outcome was the appearance of large B cell foci colocalized with DC-LAMP+, forming structures reminiscent of nascent TLS. The tumor tissue bulk RNA sequencing analysis indicated an increased expression of genes specific for B cells, DC-LAMP+ dendritic cells and FAP-CD40 induced immune activation, confirming results obtained by the Immunofluorescence analysis. Overall, this data provides robust evidence that FAP-targeted CD40 agonism to the tumor achieved a strong and sustained target engagement and tumor tissue immunomodulation, supporting further studies in combination with other anti-cancer agents in earlier lines of treatment. ***

Claims

Claims 1. A bispecific Fibroblast Activation Protein (FAP)-targeted CD40 agonistic antibody (FAP-CD40 antibody) for use in treating cancer in combination with at least one anti-cancer agent, wherein the bispecific FAP-CD40 antibody increases the density of intratumoral DC- LAMP+ DCs and B cells and activates the immune system in the tumor so that the tumor will be more sensitive to treatment with the anti-cancer agent.

2. The bispecific FAP-CD40 antibody for use of claim 1, wherein the bispecific FAP- CD40 antibody comprises at least one antigen binding domain capable of specific binding to CD40 comprising a heavy chain variable region VH (CD40) comprising the amino acid sequence of SEQ ID NO:1 and a light chain variable region VL (CD40) comprising the amino acid sequence of SEQ ID NO:2, and at least one antigen binding domain capable of specific binding to Fibroblast Activation Protein (FAP) comprising (a) a VH (FAP) comprising the amino acid sequence of SEQ ID NO:3 and a VL (FAP) comprising the amino acid sequence of SEQ ID NO:4, (b) a VH (FAP) comprising the amino acid sequence of SEQ ID NO:5 and a VL (FAP) comprising the amino acid sequence of SEQ ID NO:6, or (c) a VH (FAP) comprising the amino acid sequence of SEQ ID NO:7 and a light chain variable region VL (FAP) comprising the amino acid sequence of SEQ ID NO:

8.

3. The bispecific FAP-CD40 antibody for use of claims 1 or 2, wherein the bispecific FAP- CD40 antibody comprises a Fc domain of human IgG1 subclass with the amino acid mutations L234A, L235A and P329G (numbering according to Kabat EU index).

4. The bispecific FAP-CD40 antibody for use of any one of claims 1 to 3, wherein the bispecific FAP-CD40 antibody comprises one antigen binding domain capable of specific binding to FAP comprising the VH (FAP) comprising the amino acid sequence of SEQ ID NO:3 and the VL (FAP) comprising an amino acid sequence of SEQ ID NO:

4.

5. The bispecific FAP-CD40 antibody for use of any one of claims 1 to 5, wherein the bispecific FAP-CD40 antibody comprises a) at least two Fab fragments capable of specific binding to CD40 fused at its C-terminus to the N-terminus of a Fc region, and (b) a cross-fab fragment capable of specific binding to FAP fused to the C-terminus of the Fc region.

6. The bispecific FAP-CD40 antibody for use of claim 5, wherein the bispecific FAP- CD40 antibody comprises a cross-fab fragment capable of specific binding to FAP, wherein the VH-Ckappa chain of the cross-fab fragment is fused to the C-terminus of the Fc region.

7. The bispecific FAP-CD40 antibody for use of any one of claims 1 to 6, wherein the bispecific FAP-CD40 antibody comprises two light chains, each comprising the amino acid sequence of SEQ ID NO:9, one light chain comprising the amino acid sequence of SEQ ID NO:10, a first heavy chain comprising the amino acid sequence of SEQ ID NO:11, and a second heavy chain comprising the amino acid sequence of SEQ ID NO:

12.

8. The bispecific FAP-CD40 antibody for use of any one of claims 1 to 7, wherein the at least one anti-cancer agent is an agent blocking PD-L1 / PD-1 interaction.

9. The bispecific FAP-CD40 antibody for use of any one of claims 1 to 8, wherein the increase of the density of intratumoral DC-LAMP+ DCs and B cells is determined in a tumor biopsy by immunofluoresence (IF) analysis.

10. The bispecific FAP-CD40 antibody for use of any one of claims 1 to 8, wherein the activation of the immune system in the tumor is determined by detecting the expression level of one or more genes selected from a group consisting of NFKBID, NFKBIE, CD40, FAS, SEMA7a, CCL22, TRAF1, BCL2A1, IL1A, HSPA1A, CD83, IL4I1, CD80, CD86, CXCL10, and CFB from a tumor sample and comparing the expression level of these genes to a reference level, whereby an increase in the expression level is indicative for the activation of the immune system in the tumor.

11. The bispecific FAP-CD40 antibody for use of claims 10, wherein the activation of the immune system in the tumor is determined by detecting the expression level of all genes selected from the group consisting of FAS, SEMA7A, CCL22, TRAF1, BCL2A1, IL4I1 and CFB.

12. The bispecific FAP-CD40 antibody for use of claims 10 or 11, wherein detecting the expression level is performed by RNA sequencing analysis.

13. An in vitro method of monitoring if a patient having a tumor is responsive to cancerimmunotherapy, the method comprising determining the activation of the immune system in a tumor tissue sample obtained from the patient having received a bispecific FAP-CD40 antibody in a first treatment regimen, wherein the activation of the immune system in the tumor is characterized by detecting the expression level of all genes selected from the group consisting of FAS, SEMA7A, CCL22, TRAF1, BCL2A1, IL4I1 and CFB and wherein the method further comprises a step of comparing the expression level of the genes to a reference level, whereby anincreased expression level indicates that the tumor will be more sensitive to treatment with an anti-cancer agent in a second treatment regimen. ***

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