Inhibition of stem cell growth factor to break Anti-tumoural resistance and modulate stromal cells

Abstract

The present invention relates to antigen binding proteins that specifically bind to SCGF and comprises their use in the treatment of cancer inflammation and trauma. Also the present invention relates to nucleic acids encoding the antigen binding proteins, vectors comprising said nucleic acids and host cells comprising said expression vectors. Further, the invention relates to pharmaceutical compositions comprising said antigen binding protein and kits comprising said pharmaceutical compositions.

Description

[0001] Newl T-Amlig iarf

[0002] Deutsches •Kreteforscfcuagsze

[0003] fcWsW: Afi3105#CHJ

[0004] 1

[0005] Inhibition of stem cell growth factor to break anti-tumoural resistance and modulate stromal cells

[0006] 5 The present invention relates to an antigen binding protein that binds to Stem Cell Growth Factor (SCGF) and comprises the use of these antigen binding proteins in treating or preventing a condition associated with cancer, inflammation and / or trauma in a subject. Also, the present invention relates to nucleic acids encoding said antigen binding protein and vectors comprising the nucleic acid as well as host cells comprising the vector. Further, the invention relates to 10 pharmaceutical composition comprising said antigen-binding protein and kits comprising the pharmaceutical composition.

[0007] Background of the Invention

[0008] Known functions of Stem Cell Growth Factor (SCGF) in stem or progenitor cells

[0009] Stem Cell Growth Factor (SCGF), also known as P47, LSLCL, CLECSF3 and CLEC11A, is a secreted human growth factor belonging to the C-type lectin superfamily. It is a largely uncharacterized hematopoietic mediator that promotes progenitor cell proliferation within the bone marrow (Hiroaka et al, 2001). In humans, CLEC11 A mRNA is enriched in bone marrow (Fagerberg et al, 2014), where its expression is restricted to hematopoietic progenitor cells (Hiraka et al. 2001, 20 Perrin et al. 2001, Bannwarth et al. 1998). SCGF is a secreted cytokine that was originally defined as expressed in two isoforms: SCGFa (323 amino acids, 35,695 Da) and SCGF0 (245 amino acids, 26,902 Da) which contains a deletion within a conserved carbohydrate recognition domain (Mio et al, 1998). In combination with other hematopoietic growth factors such as granulocyte-macrophage colony- stimulating factor (GM-CSF) and erythropoietin, SCGF0 exhibits a burst-promoting activity and promotes granulocyte-macrophage colony formation (Hiraoka et al, 1997). Due to initial problems in the attribution of the sequences, SCGFa and SCGF0 protein sequences were later joined to a single sequence (from then on identified with “SCGF”).

[0010] Initially discovered on whole-body sections of embryonic mice, SCGF is expressed within skeletal tissues, particularly proliferating osteoblasts and chondroblasts, (Hiroaka et al, 2001) and within the 30 bone marrow. In the human bone marrow, SCGF is only produced by progenitor cells (CD34+and CD34 CD33+) and not mature cell types or stromal cells (Ito et al, 2003; He et al, 2005). In peripheral blood, SCGF concentration is high but drops with age (Klener et al 2013) and in adults, SCGF is present at low levels, approximately 10-20 ng / ml (Kleiner et al, 2013, Ito et al, 2003). These levels often increase along a hematopoietic recovery after bone marrow transplantation (Ito et al, 2003). Maximum levels of SCGF are observed during the rapid granulocyte recovery phase in patients subjected to an autologous transplantation. A similar response is seen in Chagas disease with idiopathic dilated cardiomyopathy. An increase in SCGF plasma levels is positively correlated with the presence of hematopoietic progenitor cells in the heart which contribute to chronic cardiac remodelling (Wang et al, 2013).

[0011] SCGF acts as an endogenous mitogen for other types of progenitor cells as well. Clara Cell Secretory Protein (CCSP, uteroglobin) is a marker for epithelial-like progenitor cells found within the bone marrow (BM) and peripheral blood (PB). In cystic fibrosis, the number of CCSP+cells in the bone marrow and peripheral blood are positively correlated with the plasma concentration of SCGF (Gilpin et al, 2013). These CCSP+cells from the BM and PB are recruited to the lung by SCGF, SDF-1 and MCP-1 where they act to reconstitute the damaged epithelial airway. This is part of a sustained effort to repair the damaged epithelium leading to a persistent inflammatory environment.

[0012] SCGF involvement in Inflammation

[0013] SCGF contributes to both localized and systemic inflammation in different diseases. In a cohort of breast cancer patients, high levels of SCGF were associated with the expression of other inflammatory cytokines such as IL-6, TNF-a and TGF-0 (Mego et al, 2016). Gundacker et al. reported the secretion of SCGF in mature pro-inflammatory dendritic cells. In spinal cord injury, SCGF and MIF (migratory inhibitory factor) contribute to secondary tissue damage which includes an inflammatory response (Ito et al, 2003). High serum levels of SCGF and MIF are also observed in pulmonary arterial hypertension (PAH), a disease in which inflammation contributes to the vascular remodelling of the precapillary pulmonary arteries (Stefanantoni et al, 2014).

[0014] In carotid artery disease with carotid plaques, activated macrophages and inflammation can lead to rupture of the plaques. Serum levels of CXCL9 and SCGF are significantly elevated in patients with unstable plaques compared to those with stable plaques. CXCL9 and SCGF increase the invasion of inflammatory cells into the plaque and contribute to their instability (Schiro et al, 2015).

[0015] In asbestos-exposed workers, SCGF increases in the serum with other inflammation markers in the early inflammatory phase (Comar et al, 2016). SCGF in Cancer

[0016] Elevated SCGF serum, gene or protein levels have been observed in many different cancer entities and SCGF is associated with aggressive, recurrent disease. It was originally detected in conditioned medium from KPB-M15 cells, a cell line derived from chronic myelogenous leukemia (CML) in blast crisis (Hiraoka et al, 1997). Accordingly, the serum levels of SCGF in CML patients is 10-100 times higher than in the disease-free controls (A. Hiraoka, 2008). In acute lymphoblastic leukemia (ALL), gene expression profiles from 35 pediatric patients demonstrated that SCGF is significantly overexpressed in recurrent disease (Bhojwani et al, 2006). In acute myelogenous leukemia (AML), CLEC11A overexpression is a predicted prognosis factor (Zhao et al 2018). Gene signatures containing SCGF were also correlated with disease relapse in solid tumours such as colorectal cancer and non-small cell lung cancer (NSCLC) (Calon et al, 2015; Lu et al, 2012). SCGF is secreted by colorectal cancer cells (cell lines Colo-205 and SW620, Wu et al 2010) and in colorectal cancer, high SCGF gene expression was detected in a more aggressive type C cancer that is characterized by a low-proliferative, chemotherapy-resistant, mesenchymal phenotype (Roepman et al, 2014). In addition, CLEC11A is reported as prognostic and immunological biomarker in gastric cancer as well as an option for clinicians to predict outcomes and formulate personalized treatment plans for gastric cancer patients (Zheng et al, 2024).

[0017] In cancer, SCGF expression is also associated with inflammation. In pleural malignant mesothelioma (PMM) patients, higher serum SCGF and IL-6 levels were observed compared to healthy volunteers. These cytokines play a role in the early pro-tumour inflammatory response (Comar et al, 2016). In gastrointestinal stromal tumours (GIST), high SCGF a was detected in protein lysates from resected tissues after treatment with imatinib (a c-KIT tyrosine kinase inhibitor). This correlated with the imatinib-induced inflammatory response that elicited monocyte / macrophage migration into the tissue (Da Riva et al, 2011).

[0018] Elevated SCGF levels have been associated with resistance to therapy. In hepatocellular carcinoma, elevated SCGF levels in the sera are found in patients who do not respond to treatment (Sukowati et al, 2018).

[0019] SCGF has been observed to promote proliferation of cancer stem cells (CSC) and maintenance of the stem cell niche. This has been observed in leukemia and some solid tumours. CSCs or tumour initiating cells are characterized by their capacity for self-renewal, multilineage differentiation, and their role in relapse and metastasis. CSCs have been identified and isolated in various cancers including leukemia, breast, brain, prostate, pancreatic, lung, and colon cancer (Reya et al, 2001). Several leukemia cell lines were shown to require self-secreted SCGF for their proliferation (A. Hiraoka, 2008). In some of these cell lines, SCGF inhibition induced apoptosis in a specific subset of cells which were identified as leukemic stem cells. They are part of a CD34 CD38 population of acute myelogenous leukemia cells that reside within the osteoblast-rich endosteal region of the bone marrow. They are able to survive chemotherapy and initiate a relapse of leukemia (Reya et al, 2001). Some data also link SCGF expression and sternness properties in solid tumours. In lung cancer, Levina et al. described that protein lysates from stem cells had high SCGF, SCF and CXCL12 levels associated with the stem cell phenotype. These cells were chemotherapy resistant in vitro and demonstrated high tumourigenic and metastatic potential in vivo.

[0020] Finally, some reports suggest that SCGF might endow tumour cells with pro-metastatic properties. In a breast cancer study, there was an observed correlation between high SCGF0 and SCF serum levels and the presence of epithelial circulating tumour cells (CTC) (Mego et al, 2016). In the ER-a-positive BG-1 ovarian carcinoma cell line in which E2 induces epithelial-to-mesenchymal transition (EMT), SCGF expression is increased after E2 treatment (Park et al, 2012).

[0021] Ovarian Cancer

[0022] Ovarian cancer is the fifth most common cancer among women worldwide with over 120,000 deaths each year (Scarlett and Conejo-Garcia, 2012). Serous ovarian carcinoma is the most common subtype and it has the highest fatality-to-incidence ratio of all gynecological malignancies (Bowtell et al, 2015). At the cellular and molecular level, ovarian cancers are very heterogeneous. So far fifteen different key oncogenes, several tumour suppressor genes and at least seven different signaling pathways have been identified that drive tumour formation and cancer progression (Bast et al, 2009). Genetic changes lead to increased proliferation, inhibition of apoptosis, blocking of anoikis, increased motility, adhesion and invasion, and attraction of stromal components adding to the complexity of the disease.

[0023] The main clinical challenge is that only 20% of women are diagnosed while the cancer is still limited to the ovaries. Most patients have advanced metastatic cancer at the time of diagnosis. For cancer in the ovary, no anatomical barrier exists to prevent widespread metastasis throughout the peritoneal cavity (Bast et al, 2009). The spread is facilitated by ascites fluid in the peritoneum that allows the passive dissemination of tumour cells, pro-tumour soluble factors and various types of immune cells (Reinartz et al, 2014). Intraperitoneal delivery of chemotherapy has improved the survival of patients and at least 70% of ovarian cancers respond to a combination of platinum and taxane based chemotherapy administered after surgery (Armstrong et al, 2006). Targeted agents like Bevacizumab (anti-VEGF antibody) also trigger some response but these treatments are failing to significantly affect overall survival. Some patients (20-40%) do not respond to first line therapy (Wang et al, 2015). Others succumb to the disease due to chemo-resistant cells that eventually lead to relapse and more widespread metastasis. Therefore, there is a great need for new therapies, which target the tumour microenvironment and can replace or add to traditional cytotoxic chemotherapy. In mice models, ovarian cancer stem cells (CSCs) have been identified. These cells are multipotent cells that can self-renew and show increased chemo-resistance to cisplatin or paclitaxel and upregulation of stem cell markers (BMI1, stem cell factor (SCF), NOTCH1, nanog, nestin and OCT4) compared with parental tumour cells grown under similar conditions (Wefers et al, 2015). These cells express protein pumps that expel chemotherapeutic drugs and so, while chemotherapy kills tumour cells, the CSCs remain and lead to recurrence. However, these cells have not been characterized well in human tumours.

[0024] Immune System involvement and potential for Immunotherapy

[0025] The immune system can play an important part in maintaining equilibrium between immune recognition and tumour development. In ovarian cancer, there is a positive correlation between the number of tumour infiltrating lymphocytes (TILs) and overall survival (Nesbeth et al, 2010, Zhang et al, 2003). The efficacy of chemotherapy, radiotherapy and antibody targeting therapy depends on interferon signalling and CD8+T cell immunity (Binder et al, 2015; Lee et al, 2009; Zitvogel et al, 2010). A transcriptome analysis of ovarian tumours showed that the presence of CD8+T cells and MHC-II molecules resulted in a better prognosis (Turner et al, 2016). Thl7 cells in the ascites produce IFN-y and IL- 17 that attract CD8+T cells to the tumour (Wefers et al, 2015). These activated T cells produce interferons that can inhibit tumour growth and IL-8 secretion, block angiogenesis and upregulate major histocompatibility complex expression increasing immune recognition (Dunn et al, 2004). This indicates that an infiltration of CD8+T-cells can prevent tumour growth if these cells are present in the tumour microenvironment.

[0026] As tumours grow, they develop mechanisms that lead to immune evasion. These mechanisms include selection of tumour variants resistant to immune effectors (sometimes designated “immune editing”), loss of target antigen expression and progressive formation of an immune suppressive environment (Vinay et al, 2015). Some subsets of immune cells also contribute to this immunosuppressive environment. In the case of ovarian cancer, this response is partly mediated by regulatory T cells (Treg), myeloid-derived suppressive cells (MDSCs) and CD16+natural killer cells (NK) (Curiel et al, 2004; Pesce et al, 2015). Dendritic cells and tumour associated macrophages (TAMs) are present in the ascites of ovarian cancer. M-CSF and MCP-1 secreted by ovarian cancer cells and by T cells are potent chemo-attractants for macrophages which produce cytokines (IL-1, IL-6 and TNF-a) that stimulate tumour growth (Bast et al, 2009). Similar to other tumour types, macrophage density in histological sections correlates with poor prognosis of ovarian cancer, highlighting the clinical relevance of TAMs (Reinartz et al, 2014). The ascites are also rich in pro-inflammatory and pro-angiogenic cytokines. For all these reasons, immunotherapy especially cellbased immunotherapy could be a promising addition to the treatment of ovarian cancer. Immune checkpoint inhibition is a form of immunotherapy that does produce long-term responses in EOC (Hamanishi et al, 2015). However, the response rates in phase II trials are too low and therefore ICB is not approved for the treatment or EOC (Zamarin et al, 2020), though research should help identify resistance mechanisms and provide a rationale for adequate co-treatment to break resistance to ICB in EOC (Kandalaft et al, 2022) and other solid tumours (Kubli et al, 2021).

[0027] Tumour Associated Macrophages (TAMs)

[0028] TAMs can promote many aspects of tumourigenesis and tumour progression, including tumour cell proliferation, invasion, angiogenesis, metastasis formation and immune suppression (Condeelis and Pollard, 2006; Sica and Bronte, 2007). Macrophages in the tumour microenvironment can be polarized to different states based on external stimuli.

[0029] Classical activation by IFN-y endows macrophages with pro- inflammatory properties mediated by the secretion of TNF-a, IL-lb, IL-6, IL-12 and IL-23. These are commonly referred to as Ml macrophages (Reinartz et al, 2014) and promote resistance to intracellular pathogens and tumours in the context of Th-1 driven responses. Alternatively, activated macrophages are elicited by antiinflammatory cytokines such as IL-4, IL- 10 or IL- 13. These are referred to as M2 macrophages and they mediate resistance to parasites, regulate, tissue repair and remodelling. This is a simplistic definition because macrophages can adopt a large variety of phenotypes deviating from this classification and may even acquire properties of both Ml and M2 cells (Mantovani et al, 2002; Qian and Pollard, 2010). The M2 macrophages, detected in human samples using CD163 marker, are often known as the pro-tumour macrophages but both types have been known to be present in the tumour microenvironment. In a cohort trial in ovarian cancer, it has been described that the presence of CD 163 positive macrophages are associated with early clinical relapse and this is unrelated to the Ml and M2 classification (Reinartz et al, 2014).

[0030] TAMs engage in complex bidirectional interactions with tumour cells, CSCs, mesenchymal stem cells (MSC) and fibroblasts. They are an essential part of the inflammatory microenvironment. Ovarian cancers show increased levels of inflammatory cytokines like TNF-a, IFN-y, IL-6, IL-8 and IL- 10, which could be secreted by the macrophages (Bast et al, 2009). TAMs can interact with CSCs and help maintain a stem cell niche through juxtacrine signalling. In breast cancer, Lu et al, found that CD90 and EphA4 are upregulated during EMT and mediate the physical interaction between CSCs and TAMs. In response, the breast cancer cells activate Src and NF-kB, which maintains the stem cell state (Lu, 2014). In hepatocellular carcinoma (HCC), the number of TAMs has a positive correlation with the density of CSCs. TAMs promote the acquisition of CSC-like properties via TGF-beta and IL-6 (Fan et al, 2014).

[0031] Cancer Associated Fibroblasts (CAFs) and Stromal Involvement

[0032] Fibroblasts are a major cellular component in the tumour microenvironment. Activated fibroblasts, also known as cancer-associated fibroblasts (CAFs), can promote tumourigenesis through immune regulation, increased angiogenesis and tumour proliferation, maintenance of sternness and inhibition of tumour cell death (Otsman and Augsten, 2009; Xing et al, 2011). CAFs can regulate the dynamic interactions between tumour cells and the extracellular matrix. They provide structural and secretory support for tumour growth and proliferation. CAFs seem to arise from multiple cell types. In some cases, they come from normal fibroblasts that transdifferentiate into activated fibroblasts through genetic and transcriptomic changes induced by the surrounding cancer cells (Xing et al, 2011). In other cases, they arise from epithelial cells through EMT, from bone marrow derived circulating cells or from mesenchymal stem cells (Anderberg and Pietras, 2009).

[0033] The functions of CAFs have been described in all stages of tumour development, from initiation to metastasis outgrowth. Fibroblast activation protein (FAP), also known as FAPa or separase has emerged as an important marker of CAFs in cancer (Mhawech-Fauceglia et al, 2014). FAPa is not typically expressed in normal tissue, but is expressed by fibroblasts in wound healing and on mesenchymal stem cells derived from human bone marrow (Bae et al, 2008). FAPa is highly expressed in activated fibroblasts in more than 90% of human epithelial carcinomas, including pancreatic, hepatocellular, breast, lung, colorectal and ovarian cancers (Lai et al, 2012). It is also upregulated in CAF precursor cells that are recruited to the growing tumour (Iwasa et al, 2005). In ovarian cancer, tumour-associated stromal expression of FAPa has been found to be associated with more aggressive disease progression and plays an important role in chemotherapy response, via cell autonomous and non-cell autonomous mechanisms (Mhawech-Fauceglia et al, 2014). CAFs shape drug resistance. FAPa positive CAFs were indeed shown to reduce cisplatin accumulation in ovarian cancer cells (Wang et al, 2015).

[0034] Another important function of CAFs is in promoting inflammation. Ovarian and breast carcinomas are amongst the leading causes of cancer-related mortality in women and inflammation is linked with both these tumour types. CAFs in these cancer types express high levels of IL-6, COX-2 and CXCL12 which are part of a pro-inflammatory gene signature. The pro-inflammatory genes expressed by CAFs are known NF-KB targets and NF-KB is up-regulated in breast and ovarian CAFs (Erez et al, 2013).

[0035] CAFs can also regulate the CSC niche. Ovarian cancer FAPa positive CAFs were analyzed with qRT-PCR and they showed expression of Nanog, Oct-4, Sox2, nestin, CD133 and hTERT indicating that CAFs show some sternness themselves. In prostate cancer, CAF-induced EMT leads to the enhanced expression of stem cell markers and increases the ability of these cells to form prostaspheres and to self-renew (Giannoni et al, 2010). Vermeulen et al. reported that primary isolated colon CAFs release HGF that induced a nuclear translocation of beta-catenin in tumour cells thereby inducing a stem cell typical gene transcription profile (Weiland et al, 2012). CAFs play a central role in regulating CSC phenotypes by providing regulatory factors such as HGF, IL-6, chemokines and by regulating the Wnt and TGF-beta pathways (Weiland et al, 2012). Thus, the paracrine interplay between CAFs and cancer cells leads to the maintenance of cancer stem cell properties associated with metastasis and tumour relapse.

[0036] Finally, CAFs are inhibitory to the establishment of an anti -tumour immune response through their immunosuppressive cytokine secretion (eg. CXCL12) (Kraman et al, 2010) and through the establishment of a thick collagen wall at the tumour-stroma interface that is impenetrable and leads to immune exclusion (Sun et al, 2021).

[0037] Therapeutic Antibodies

[0038] Antibody-based therapy for cancer has become established over the past 15 years and is now an important strategy for treating patients with both haematological malignancies and solid tumours. There are a number of antigens that are expressed by human tumours, which could be targets for antibody-based therapy (Chames et al. 2009). These antibodies work by mediating alterations in antigen or receptor function, modulating the immune system, causing vascular or stromal cell ablation or delivering a specific drug coupled to the antibody (Chames et al, 2009). The successful development of candidate antibodies involves a complex process of evaluation that includes identification of the physical and chemical properties of the antibody, analysis of antigen presentation, study of effector functions and signalling pathways of the antibody, in vivo localization, distribution and therapeutic activity (Scott et al, 2012). It is vital to assess the ratio of antibody uptake in the tumour versus normal tissues to avoid unwanted side effects. By 2014, more than 30 monoclonal antibodies had been approved by the FDA for use in different cancers and more than 240 therapeutic monoclonal antibodies are under clinical trials (Ju and Jung, 2014). The number of antibodies getting first approval always gets higher (3 approved in 2019, 6 in 2022) (https: / / www.antibodysociety.org / resources / approved-antibodies).

[0039] IgG antibodies are glycoproteins with two main regions, the Fab region and Fc region. The Fab is the antigen binding domain. It is composed of one constant and one variable domain that each contain a light and heavy chain. The variable region recognizes the antigen and binds to it. The Fc region is composed of heavy chains and this region mediates binding with immune effector cells.

[0040] Antibody Engineering: Chimeric Antibodies

[0041] Historically, therapeutic antibodies were initially produced in mice but the murine antibodies were rejected by the human immune system. A major breakthrough in antibody engineering was the production of chimeric antibodies. Chimeric antibodies are 70% human and possess a fully human Fc portion which makes them less immunogenic in humans and allows interaction with immune cell elements (Chames et al. 2009). In chimeric antibodies, the murine variable domains are fused with human constant domains. The binding activity of IgG molecules is generated by the variable domains of the heavy and the light chains therefore the murine variable domains will still bind to the antigen.

[0042] Antibody uptake by the tumour depends on efficient penetration and retention in the targeted tissue and characteristics such as molecular size, shape and affinity control these properties (Chames et al, 2009). The Fc portion of IgGs can interact with the neonatal Fc receptor (FcRn) expressed at the surface of several cell types which increases their retention in circulation. IgG antibodies engage the cellular immune system via interaction with of the Fc domain through Fey receptors (FcyRs) (Nimmerjahn and Ravetch, 2007). The human FcyR family contains six known members in three subgroups, including FcyRI (CD64), FcyRIIa,b,c (CD32a,b,c) and FcyRIIIa,b (CD16a,b), expressed by various effector cells of the immune system, including macrophages, neutrophils, dendritic cells and natural killer (NK) cells. These cells interact with the IgG molecule at an N-linked glycosylation domain at N297 in the CH2 domain of the Fc region. This interaction activates antibody-dependent cell-mediated toxicity (ADCC), antibody dependent cell mediated phagocytosis (ADCP) and complement dependent cytotoxicity (CDC) (Ju and Jung, 2014) which are essential for destroying antigens.

[0043] There are some limitations to the use of chimeric antibodies for therapy. FcyR polymorphism make it difficult to predict clinical responses to antibody therapy (DiLillo and Ravetch 2015). This makes it difficult to assess the exact in vivo affinity of the antibody and its antigen because the therapeutic antibody has to compete with a high concentration of patient’s IgGs for binding to FcyRIIIa. There are also inhibitory Fc receptors that reduce antibody activity such as FcyRIIb, expressed by B-cells, macrophages, dendritic cells and neutrophils (Nimmerjahn and Ravetch, 2007). Another limiting factor is tissue penetration, especially in the case of solid tumours. Few approved antibodies target solid tumours but over 85% of human cancers are solid tumours, clearly reflecting a limitation of antibody treatment.

[0044] Antibody Engineering: Single chain variable fragment (scFv) antibodies

[0045] Apart from making full length antibodies, it is also possible to make versions that are smaller and address some of the limitations found in the full length chimeric antibodies. Single chain variable fragments (scFv) are small fragments capable of retaining the binding activity of the full IgG molecule (Bird et al, 1988). They contain the variable domains of the heavy and light chains linked by a flexible linker. Because of the very short half-life in serum (~2 h), this fragment has less overall tumour uptake. However, due to their reduced size, antibody fragments usually penetrate tumours much more rapidly and efficiently than full IgG.

[0046] Summary of the Invention

[0047] In a first aspect the present invention provides an antigen binding protein that specifically binds to Stem Cell Growth Factor (SCGF), wherein said antigen binding protein competes for binding to SCGF with a reference antibody comprising a heavy chain variable region of the amino acid sequence in SEQ ID NO: 1; and a light chain variable region of the amino acid sequence in SEQ ID NO: 2.

[0048] In a second aspect the present invention provides an antigen binding protein that specifically binds to SCGF, wherein said antigen binding protein comprises either:

[0049] (i) a combination of a light chain variable domain and a heavy chain variable domain selected from the group of combinations consisting of: a heavy chain variable domain having a sequence with at least 90% identity to SEQ ID NO: 1; and

[0050] a light chain variable domain having a sequence with at least 90% identity to SEQ ID NO: 2 and

[0051] (ii) a complementarity determining region 3 of the heavy chain (CDRH3) comprising or consisting of the amino acid sequence of SEQ ID NO: 5 and a complementarity determining region 3 of the light chain (CDRL3) comprising or consisting of the amino acid sequence of SEQ ID NO: 8.

[0052] In a third aspect the present invention provides an antigen binding protein that specifically binds to Stem Cell Growth Factor (SCGF), wherein said antigen binding protein comprises

[0053] a CDRH1 comprising or consisting of the amino acid sequence of SEQ ID NO: 3, a CDRH2 comprising or consisting of the amino acid sequence of SEQ ID NO: 4, a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 5, a CDRL1 comprising or consisting of the amino acid sequence of SEQ ID NO: 6, a CDRL2 comprising or consisting of the amino acid sequence of SEQ ID NO: 7, and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 8; and / or

[0054] binds to an epitope of SCGF that comprises or consists of the amino acid sequence PVWLGVHD (SEQ ID NO: 11) of SEQ ID NO: 9:

[0055] In a fourth aspect the present invention provides a nucleic acid or a set of two nucleic acid molecules encoding the antigen binding protein of the first, second or third aspect of the invention.

[0056] In a fifth aspect the present invention provides a recombinant expression vector or a set of two recombinant expression vectors comprising a nucleic acid molecule(s) according to the fourth aspect of the present invention.

[0057] In a sixth aspect the present invention provides a host cell comprising the vector of the fifth aspect of the invention.

[0058] In a seventh aspect the present invention provides a method of making the antigen binding protein of the first, second or third aspect of the invention, comprising the step of preparing said antigen binding protein from a host cell expressing said antigen binding protein.

[0059] In an eighth aspect the present invention provides a pharmaceutical composition comprising at least one antigen binding protein according to the first, second or third aspect of the invention, the nucleic acid according to the fourth aspect of the invention, or the vector according to the fifth aspect of the invention and optionally further comprising a pharmaceutically acceptable excipient. In a ninth aspect the present invention provides a kit for the treatment of cancer, inflammation, or trauma comprising the pharmaceutical composition of the eighth aspect of the invention.

[0060] In an tenth aspect the present invention provides a SCGF antagonist for use in treating or preventing a condition associated with cancer, inflammation and / or trauma in a subject.

[0061] In an eleventh aspect the present invention provides at least one antigen binding protein according to first, second or third aspect of the invention, the nucleic acid according to the fourth aspect of the invention, the vector according to the fifth aspect of the invention, the host cell of the sixth aspect of the invention, the pharmaceutical composition of the eighth aspect of the invention, or a combination thereof and optionally in addition one of the further compounds being selected from items (i) to (iv) of the preferred embodiment of the eight aspect for use in treating or preventing a disease, wherein the disease is preferably a cancer, an inflammatory disease and / or a trauma in a subject, wherein the subject is preferably an adult.

[0062] List of Figures

[0063] In the following, the content of the figures comprised in this specification is described. In this context please also refer to the detailed description of the invention above and / or below.

[0064] Figure 1: (A) Schematic representation of a human IgG antibody with the Fab and Fc-binding region. (B) Full length chimeric antibodies are produced by combining the mouse Fab variable region linked to human IgG molecules. The heavy and light chains of the variable regions connected by a linker make up the scFv fragment (modified from absolute antibody, online).

[0065] Figure 2: An overview of the Luminex xMAP technology used to measure cytokine levels. The capture beads are magnetic beads coupled to monoclonal antibodies from 50 different cytokines. The detection antibodies are biotinylated with a Streptavidin-Phycoerythrin (SA-PE) reporter that is excited by the green laser and is detected by fluorescence (Bio-Rad).

[0066] Figure 3: The cDNA sequence used to clone our antibodies (SEQ ID NO: 16). The heavy and light chains are linked with a glycine rich linker. This entire sequence was the scFv fragment that was ligated to human IgGl (CH2-CH3) (referred to herein as scFv-hlgGl) or mouse IgG2a (referred to herein as scFv-mIgG2a). For the full length antibody, the individual heavy and light chains were cut out and ligated to human IgGl (CH1-CH2-CH3) and human Ig kappa respectively. There were two plasmids for the full length antibody.

[0067] Figure 4: SDS Page gels confirmed the size of the purified antibodies: In both gels, the supernatant from the transfection (CR), the flow-through from the column (FT) and the wash pool (WP) were run under reducing conditions. Five micrograms of the highest elution fraction was run under reducing and non- reducing (NR) conditions. The picture was taken after staining the gels with instantBlue. (A) scFv-mIgG2a and scFv-hIgG1 antibodies produced in CHO-S cells after protein A purification were run after transfection in CHO-S cells and purified using protein A. The purified antibodies showed distinct bands at around 50KDa (52KDa was the expected size for these antibodies). (B) Full length antibody produced by double transfection in both CHO-S cells and HEK cells and purified with protein A. Two distinct bands were observed around 50KDa and 30KDa. The light chain was expected at 23.5KDa and the heavy chain was expected at 52KDa.

[0068] Figure 5: Characterization of the binding and binding sites of the anti-SCGF antibody. (A) A capture ELISA with recombinant SCGF confirmed antigen binding of the purified antibodies compared to the hybridoma supernatant: All the purified antibodies were tested at three different concentrations along with non-purified hybridoma supernatant. (B) Illustration of the epitope mapping results. The binding of the scFv-mIgG2a was tested on a series of overlapping peptides (15mers) encompassing the whole sequence of recombinant SCGF, with an offset of 4 amino acids between each peptide.

[0069] Figure 6: The scFv-mIgG2a anti-SCGF antibody produces staining patterns in IHC that are similar to the hybridoma supernatant: immunostaining for SCGF using either hybridoma supernatant (1:200) or scFv-mIgG2a (1:500) on serial cryosections from EOC.

[0070] Figure 7: anti-SCGF treatment in tumour explants show modulation in different cytokines after 48 hours. Two EOC explant cultures were treated with hybridoma supernatant (1:200) and the scFv-hIgG1 anti-SCGF antibodies in two different concentrations (2.5µg / ml and 5µg / ml), prior protein lysis and multiplex cytokine measurement. After 48 hours in each tissue treated, the patterns were similar in the tissue sections treated with hybridoma supernatant and the scFv-hIgG1 anti-SCGF antibody.

[0071] Figure 8: SCGF levels do not correlate to increased angiogenesis or immune cell infiltration. Numbers of (A) CD105+tumour-associated blood vessels, (B) CD8+cytotoxic T cells, (C) CD3+T cells and (D) CD163+TAMs per square millimetre detected by immunostaining and quantified using Visiopharm on the whole slide image. These numbers are plot across the corresponding SCGF concentration obtained from multiplex cytokine measurement.

[0072] Figure 9: Macrophages from breast cancer ascites express SCGF. Representative illustration of an IHC stain for SCGF (using the hybridoma supernatant) on macrophages isolated from a breast cancer-associated ascites. Figure 10: Immunofluorescent staining reveals expression of SCGF in tumour associated macrophages. Macrophages from breast cancer ascites were double positive for CLEC5A and SCGF or CD 163 and SCGF. The scale bar is at 100pm. The nuclei were stained with DAPI.

[0073] Figure 11: FAP+cancer associated fibroblasts might be expressing SCGF in the stroma of ovarian cancer tissues: (A) representative image of immunostaining for SCGF (left) and FAPalpha (right) in an ovarian carcinoma tissue. The staining patterns of FAPalpha and SCGF appear similar. Ovarian cancer tissues show high expression of FAPalpha and SCGF seems to be localized in fibroblastic cells. (B) A virtual overlay of anti-CD68, anti-SCGF and anti-FAPalpha immunostainings on consecutive cryosections from the same tissue. (C) Double immunofluorescent stain for SCGF and FAPalpha on a cryosection of an EOC specimen, confirming the localization of SCGF in FAPalpha cells.

[0074] Figure 12: Restriction map of vectors A) pcDNA 3.1 and B) pBluescript KSII+

[0075] Figure 13: Illustration of Luminex quantification results as expressed by score distribution in 40 tissue culture experiments and examples of tissues responding or not to anti-SCGF treatment.

[0076] Figure 14:_Anti-SCGF triggers T cell and CD8+ cytotoxic T cell expansion in the tumour in 75% of treated explants (A) CD3 immunostaining explants cultured for 48h with anti-SCGF and (B) microimage at a higher magnification, with the corresponding quantification of the whole image.

[0077] (C) Pan T cell (CD3), cytotoxic T cell (CD8) and activated T cell (Granzyme B) numbers in explants treated with anti-SCGF, compared to serial explants left untreated. (C) Response rate in explants to either anti-SCGF or Nivolumab and corresponding response rates in a phase II trial.

[0078] Figure 15: Anti-SCGF treatment consistently leads to a molecular signature of macrophage repolarization and of the activation of the Thl / cytotoxic anti-tumour immune response. (A) Example of one response to anti-SCGF in an EOC explant culture exhibiting macrophage repolarization. (B) Example of one response to anti-SCGF in a tissue exhibiting activated Thl / cytotoxic immune response. (C) Concentration of three factors involved in T cell survival and chemotaxis and their modulation by anti-SCGF in 40 EOC explant culture. (D) Scores of the response to anti-SCGF in explants from diverse solid tumours cultured in the laboratory. MMMT: malignant mixed mullerian tumour (also known as uterine carcinosarcoma). CUP: cancer of unknown primary. (E) Method to generate the score of response to anti-SCGF. (F) Effects of anti-SCGF on the cytokine secretions in TAMs isolated from malignant ascites.

[0079] Figure 16: the effects induced by anti-SCGF treatment are Fab-dependent. (A) In an EOC explant culture, the molecular effects induced by anti-SCGF treatment are not induced by the treatment with a negative control antibody with the same structure (one representative experiment out of 5 is shown as an example). (B) The dot plot illustrates the molecular changes induced in cell culture with anti-SCGF treatment overnight (each dot is a cytokine and chemokine from the Luminex protein measurement). Monocytes were isolated from a healthy donor and either treated as such, or differentiated into macrophages or into tumour-associates macrophages in vitro prior treatment. One representative experiment out of two is shown.

[0080] Figure 17: anti-SCGF treatment kills tumour cells in tissue explant cultures from solid tumours. (A) Microimages illustrating the EpCAM-activated caspase 3 double immunostaining for the detection of apoptotic tumour cells and its automated detection with the Halo virtual pathology software. (B) Proportion of dying tumour cells (as measured by EpCAM+active caspase3+cells / total EpCAM+cells) in explants from EOC and from one CUP. (C) In one explant from a stomach cancer, quantification of the activated caspase 3 single immunostaining. The analysis was restricted to epithelial tumour islets.

[0081] Figure 18: SCGF levels associate with a network of densely aligned collagen fibres in EOC.

[0082] (A) Masson trichrome stain in EOC explants after anti-SCGF treatment and (B) second harmonics generation imaging analysis enabling the direct visualization and quantification of collagen fibres, as well as (C) corresponding quantification. Images were acquired at 40X magnification. From the raw data, fibre segmentation in CurveAlign / CT-Fire and quantitative assessment of fibre structure, fibre width, length and relative angle of approximately n = 2000 recorded events in each group. (D) Masson trichrome stains of EOC specimens exhibiting SCGFhigh(here: above 10.000 pg / ml) or SCGFlow(under 2.000 pg / ml)) concentration. (E) Second harmonics generation imaging of SCGFhighand SCGFlowtissues.

[0083] Figure 19: Effects of anti-SCGF treatment in EOC explants pre-treated with clodronate liposomes. (A) Masson trichrome images from explants treated with anti-SCGF and pre-treated with clodronate liposomes (or PBS liposomes as a vehicle control) to inhibit the macrophage functions.

[0084] (B) Number of T cells in whole slide image analysis of n = 9 patient-derived EOC tissue cultures treated as in (A) and showing a decrease in T cell numbers in the tumour in case of clodronate liposome pretreatment.

[0085] Figure 20: proposed mode-of-action of anti-SCGF antibodies as immunotherapy in solid tumours.

[0086] Figure 21: efficacy of anti-SCGF treatment in solid tumours beyond EOC. (A) Microimages of immunostaining for SCGF (carried out with the scFv-mIgG2a as primary antibody) in various solid tumours showing consistent accumulation of SCGF in the stroma areas. (B) Concentration of SCGF in the tumour lysates in multiple solid tumours as measured by Luminex-based protein quantification. (C) Representative cases of response to anti-SCGF treatment with increased CD8+cytotoxic cell numbers in solid tumours and (D) with increased apoptotic cancer cell death.

[0087] Figure 22: SCGF levels increase in tumour explants upon treatment with respective standard chemotherapy in various tumour entities. (A) SCGF levels in the tumour lysates of EOC samples received from patients who were either treatment-naive or who had received chemotherapy already.

[0088] (B) SCGF levels in EOC explants increased after treatment with carboplatin, (C) this increases is directly dependent on the concentration or carboplatin. (D) SCGF levels in liver metastases from colorectal cancer after explant culture with oxaliplatin. (E) SCGF levels in explant cultures of pancreatic cancer after treatment with FOLFIRI (folinic acid, fluorouracil and irinotecan).

[0089] Figure 23: Inhibition of hepatocyte growth factor breaks resistance to anti-SCGF in EOC.

[0090] (A) HGF concentration in EOC tumour lysates according to their concentration of SCGF. (B) HGF concentration in EOC explants according to whether or not they respond to anti-SCGF treatment with T cell expansion (R = responders, NR = non-responders). (C) Synergistic response to anti-SCGF and Capmatinib-mediated HGF receptor inhibition at the molecular level. (D) CD8+cytotoxic T cell stains in EOC explants treated with anti-SCGF and Capmatinib and (E) corresponding quantification.

[0091] Figure 24: Anti-SCGF breaks resistance to adoptive cell therapy. (A) Number of total CD8+cytotoxic T cells in explants from a lung metastasis treated with autologous TILs and anti-SCGF for 24 hours. (B) Number of CMFDA-labelled, non-autologous CAR T cells infiltrating a primary colorectal cancer explants after 24 hours of culture with anti-SCGF treatment.

[0092] Figure 25: Anti-SCGF treatment synergizes with immune checkpoint blockade (Nivolumab treatment). (A-C) Increased SCGF concentration is observed after treatment with Nivolumab in explant cultures of EOC (A), malignant melanoma (B) and colorectal cancer-liver metastases (C). (D) In EOC, one case of synergistic expansion of CD8+cytotoxic T cells was observed after simultaneous treatment with anti-SCGF and Nivolumab. (E) In two other EOC explant cultures, a synergistic induction of cancer cell death was observed after double treatment. The effects observed in (E) are independent of T cell numbers (F).

[0093] Figure 26: Anti-SCGF and maraviroc treatment synergize in solid tumours. (A) Example of an EOC tissue culture where no response to either anti-SCGF or to maraviroc is observed, yet the characteristic molecular response is achieved after simultaneous treatment with anti-SCGF and maraviroc. (B) Corresponding CD8+cytotoxic T cell numbers indicating the synergy between anti-SCGF and maraviroc also applies to T cell expansion. (C) Example of a stomach cancer growing in the ovaries, where a similar synergy as in (A) is observed. Figure 27: Cancer cells respond to exposure to SCGF with increased proliferation and survival. (A) Cell counts of primary epithelial ovarian cancer cells exposed to recombinant SCGF for 24h. (B) Cell counts of an EOC cell line (SKOV3) exposed to recombinant SCGF for 24h. (C) Cell counts of two liver metastasis cell lines exposed to recombinant SCGF for 72h. (D) Proportion of dead cells (as measured by counting Trypan blue-positive cells) in a culture of primary EOC cells exposed to recombinant SCGF for 24h.

[0094] Figure 28: SCGF expression and signalling pathways in the tumour environment. (A) anti-SCGF treatment in TAMs decreases phosphorylation of the CREB transcription factor and (B) elevated SCGF levels in the whole tumour lysates are associated with decreased phosphorylation levels of CREB and c-Jun.

[0095] Figure 29: Modulation of cytokine secretion by recombinant stem cell growth factor (SCGF) or anti-SCGF antibodies. Differentiated human monocytic THP-1 cells were treated with indicated concentrations of recombinant (rec.) SCGF or anti-SCGF antibodies and stimulated by bacterial lipopolysaccharides (LPS). Secretion of indicated cytokines was determined in cell culture supernatants after 12 h by ELISA and indicated as percent change from stimulation alone. A) Scheme of the experiment. B, C) Change of TNF secretion by differentiated THP-1 cells treated with indicated antibodies and stimulated by LPS, relative to cells stimulated by LPS only. D) Change of IL-1ß-secretion by differentiated THP-1 cells treated with indicated agents (rec. SCGF [10 ng / ml] or different antibodies as indicated) and stimulated by LPS [100 ng / ml], relative to cells stimulated by LPS only. 8326.1 = anti-SCGF antibody developed by DKFZ; ProTech = anti-SCGF antibody sold by Proteintech Germany GmbH; R& D = anti-SCGF antibody sold by R& D Systems.

[0096] Detailed Descriptions of the Invention

[0097] It can be taken from the appended examples, in particular the section “Concepts and evidence behind invention” that the exemplified antigen binding protein specifically binds to SCGF precisely on the C-lectin-type domain (Figure 5). The antigen binding protein neutralizes the binding to the C-lectin-type domain of SCGF. It is also shown that the binding to the C-lectin-type domain effect the expression of a plethora of cytokines, thereby enabling the effective use of the antigen binding protein for various cancer types. The examples show that the cytokines affected by SCGF inhibition cancer tissue culture can be grouped into different categories based on their functions. SCGF inhibition results in an increase in pro-inflammatory, Thl supportive cytokines that promote the activation of an anti-tumour adaptive immune response. Also, an increase in cytotoxic factors is observed and finally, anti-SCGF triggers an increase in a multitude of chemokines that boost intra- tumour infiltration by immune cells as well as factors supportive of T cell survival. In some tissues, a decrease in pro-angiogenic factors can also be observed. These changes were accompanied by the proliferation and expansion of T cells and CD8+cytotoxic T cells that were activated (Granzyme B+) and therefore capable of killing tumour cells. The results indicated as response in 75 % of the treated tissues. The anti-SCGF antibody of the invention in particular outperformed the commercially available anti-SCGF antibodies from Proteintech (60-295-I-Ig) and R& D (MAB1904). The activating effect on macrophages, enhancing pro-inflammatory cytokines (TNFa and IL-1ß) was enhanced by up to 25% by the addition of the antibody 8326.1 in a dose dependent fashion, while a less pronounced effect was observed for Proteintech (60-295-I-Ig), and only a minor effect was observed for R& D (MAB1904) (Figure 29). Hence, the data in the appended examples demonstrate that the anti-SCGF antigen binding protein as provided herein is particularly well suitable and better suitable than the tested commercial anti-SCGF antibody of the prior art for the treatment and prevention of diseases, such as cancer.

[0098] Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0099] Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. Some of the documents cited herein are characterized as being “incorporated by reference”. In the event of a conflict between the definitions or teachings of such incorporated references and definitions or teachings recited in the present specification, the text of the present specification takes precedence.

[0100] In the following, the elements of the present invention will be described. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.

[0101] Definitions

[0102] In the following, some definitions of terms frequently used in this specification are provided. These terms will, in each instance of its use, in the remainder of the specification have the respectively defined meaning and preferred meanings.

[0103] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents, unless the content clearly dictates otherwise.

[0104] As used in this specification the term “nucleic acid” comprises polymeric or oligomeric macromolecules, or large biological molecules, essential for all known forms of life. Nucleic acids, which include DNA (deoxyribonucleic acid) and RNA (ribonucleic acid), are made from monomers known as nucleotides. Most naturally occurring DNA molecules consist of two complementary biopolymer strands coiled around each other to form a double helix. The DNA strand is also known as polynucleotides consisting of nucleotides. Each nucleotide is composed of a nitrogen-containing nucleobase as well as a monosaccharide sugar called deoxyribose or ribose and a phosphate group. Naturally occurring nucleobases comprise guanine (G), adenine (A), thymine (T), uracil (U) or cytosine (C). The nucleotides are joined to one another in a chain by covalent bonds between the sugar of one nucleotide and the phosphate of the next, resulting in an alternating sugar-phosphate backbone. If the sugar is desoxyribose, the polymer is DNA. If the sugar is ribose, the polymer is RNA. Typically, a polynucleotide is formed through phosphodiester bonds between the individual nucleotide monomers. In the context of the present invention the term “nucleic acid” includes but is not limited to ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and mixtures thereof such as e.g. RNA-DNA hybrids (within one strand), as well as cDNA, genomic DNA, recombinant DNA, cRNA and mRNA. A nucleic acid may consist of an entire gene, or a portion thereof, the nucleic acid may also be a miRNA, siRNA, or a piRNA. MiRNAs are short ribonucleic acid (RNA) molecules, which are on average 22 nucleotides long but may be longer and which are found in all eukaryotic cells, i.e. in plants, animals, and some viruses, which functions in transcriptional and post-transcriptional regulation of gene expression. MiRNAs are post-transcriptional regulators that bind to complementary sequences on target messenger RNA transcripts (mRNAs), usually resulting in translational repression and gene silencing. Small interfering RNAs (siRNAs), sometimes known as short interfering RNA or silencing RNA, are short ribonucleic acid (RNA molecules), between 20-25 nucleotides in length. They are involved in the RNA interference (RNAi) pathway, where they interfere with the expression of specific genes. PiRNAs are also short RNAs which usually comprise 26-31 nucleotides and derive their name from so-called piwi proteins they are binding to. The nucleic acid can also be an artificial nucleic acid. Artificial nucleic acids include polyamide or peptide nucleic acid (PNA), morpholino and locked nucleic acid (LNA), as well as glycol nucleic acid (GNA) and threose nucleic acid (TNA). Each of these is distinguished from naturally- occurring DNA or RNA by changes to the backbone of the molecule.

[0105] The term "sample" is referring to a part or piece of a tissue, organ or individual, typically being smaller than such tissue, organ or individual, intended to represent the whole of the tissue, organ or individual. Upon analysis a sample provides information about the tissue status or the health or diseased status of an organ or individual. Examples of samples include but are not limited to fluid samples such as blood, serum, plasma, synovial fluid, lymphatic fluid, cerebrospinal fluid, meningeal fluid, glandular fluid, fine needle aspirate, spinal fluid and other body fluids (urine, saliva). Further examples of samples include cell cultures or tissue cultures. Further examples include as well liquid and solid biopsy samples or solid samples such as tissue extracts. Samples may comprise fossils, remnants from extinct organisms, plants, fruits, animals, microbes, bacteria, viruses, fungi or cells derived therefrom.

[0106] The term "vector" is referring to any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage or virus) used to transfer protein coding information into a host cell.

[0107] The term "expression vector" or "expression construct" refers to a vector that is suitable for transformation of a host cell and contains nucleic acid sequences that direct and / or control (in conjunction with the host cell) expression of one or more heterologous coding regions operatively linked thereto. An expression construct can include, but is not limited to, sequences that affect or control transcription, translation, and, if introns are present, affect RNA splicing of a coding region operably linked thereto.

[0108] The term "operably linked" as used herein, means that the components to which the term is applied are in a relationship that allows them to carry out their inherent functions under suitable conditions. For example, a promoter sequence in a vector that is "operably linked" to a protein coding sequence is ligated thereto so that expression of the protein coding sequence is achieved under conditions compatible with the transcriptional activity of the promoter sequences.

[0109] The term "host cell" is referring to a cell that has been transformed, or is capable of being transformed, with a nucleic acid sequence and thereby expresses a gene of interest. The term includes the progeny of the parent cell, whether or not the progeny is identical in morphology or in genetic make-up to the original parent cell, so long as the gene of interest is present.

[0110] The terms "polypeptide" or "protein" are referring to a macromolecule having the amino acid sequence of a native protein, that is, a protein produced by a naturally- occurring and nonrecombinant cell; or it is produced by a genetically-engineered or recombinant cell, and comprise molecules having the amino acid sequence of the native protein, or molecules having deletions from, additions to, and / or substitutions of one or more amino acids of the native sequence. The term also includes amino acid polymers in which one or more amino acids are chemical analogs of a corresponding naturally-occurring amino acid and polymers. The terms "polypeptide" and "protein" specifically encompass SCGF antigen binding proteins, antibodies, or sequences that have deletions from, additions to, and / or substitutions of one or more amino acid of antigen-binding protein. The term "polypeptide fragment" refers to a polypeptide that has an amino-terminal deletion, a carboxyl-terminal deletion, and / or an internal deletion as compared with the full-length native protein. Such fragments can also contain modified amino acids as compared with the native protein. In certain embodiments, fragments are about five to 500 amino acids long. For example, fragments can be at least 5, 6, 8, 10, 14, 20, 50, 70, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acids long. Useful polypeptide fragments include immunologically functional fragments of antibodies, including binding domains. In the case of a SCGF-binding antibody, useful fragments include but are not limited to a CDR region, a variable domain of a heavy and / or light chain, a portion of an antibody chain or just its variable region including two CDRs, and the like.

[0111] The term "isolated protein" refers to a subject protein that (1) is free of at least some other proteins with which it would normally be found, (2) is essentially free of other proteins from the same source, e.g. from the same species, (3) is expressed by a cell from a different species, (4) has been separated from at least about 50 percent of polynucleotides, lipids, carbohydrates, or other materials with which it is associated in nature, (5) is operably associated (by covalent or noncovalent interaction) with a polypeptide with which it is not associated in nature, or (6) does not occur in nature. Typically, an "isolated protein" constitutes at least about 5%, at least about 10%, at least about 25%, or at least about 50% of a given sample. Genomic DNA, cDNA, mRNA or other RNA, of synthetic origin, or any combination thereof can encode such an isolated protein. Preferably, the isolated protein is substantially free from proteins or polypeptides or other contaminants that are found in its natural environment that would interfere with its therapeutic, diagnostic, prophylactic, research or other use.

[0112] The term "amino acid" includes its normal meaning in the art. 1

[0113] A "variant" of a polypeptide (e.g. an antigen binding protein, or an antibody) comprises an amino acid sequence wherein one or more amino acid residues are inserted into, deleted from and / or substituted into the amino acid sequence relative to another polypeptide sequence. Variants include fusion proteins.

[0114] The term "identity" refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. " Percent identity" means the percent of identical residues between the amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest of the molecules being compared. For these calculations, gaps in alignments (if any) are preferably addressed by a particular mathematical model or computer program (i.e., an "algorithm"). Methods that can be used to calculate the identity of the aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, A. M., ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, D. W., ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, A. M., and Griffin, H. G., eds.), 1994, New Jersey: Humana Press; von Heinje, G, 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math. 48:1073.

[0115] In calculating percent identity, the sequences being compared are typically aligned in a way that gives the largest match between the sequences. One example of a computer program that can be used to determine percent identity is the GCG program package, which includes GAP (Devereux et al., 1984, Nucl. Acid Res. 12:387; Genetics Computer Group, University of Wisconsin, Madison, WI). The computer algorithm GAP is used to align the two polypeptides or polynucleotides for which the percent sequence identity is to be determined. The sequences are aligned for optimal matching of their respective amino acid or nucleotide (the "matched span", as determined by the algorithm). A gap opening penalty (which is calculated as 3x the average diagonal, wherein the "average diagonal" is the average of the diagonal of the comparison matrix being used; the "diagonal" is the score or number assigned to each perfect amino acid match by the particular comparison matrix) and a gap extension penalty (which is usually 1 / 10 times the gap opening penalty), as well as a comparison matrix such as PAM 250 or BLOSUM 62 are used in conjunction with the algorithm. In certain embodiments, a standard comparison matrix (see Dayhoff et al., 1978, Atlas of Protein Sequence and Structure 5:345-352 for the PAM 250 comparison matrix; Henikoff et al., 1992, Proc. Natl. Acad. Sci. U. S. A. 89:10915-10919 for the BLOSUM 62 comparison matrix) is also used by the algorithm.

[0116] Examples of parameters that can be employed in determining percent identity for polypeptides or nucleotide sequences using the GAP program are the following:

[0117] Algorithm: Needleman et al., 1970, J. Mol. Biol. 48:443-453

[0118] Comparison matrix: BLOSUM 62 from Henikoffet al., 1992, supra

[0119] Gap Penalty: 12 (but with no penalty for end gaps)

[0120] Gap Length Penalty: 4

[0121] Threshold of Similarity: 0

[0122] Certain alignment schemes for aligning two amino acid sequences may result in matching of only a short region of the two sequences, and this small aligned region may have very high sequence identity even though there is no significant relationship between the two full-length sequences. Accordingly, the selected alignment method (GAP program) can be adjusted if so desired to result in an alignment that spans at least 50 or other number of contiguous amino acids of the target polypeptide.

[0123] As used herein, the twenty conventional (e.g. naturally occurring) amino acids and their abbreviations follow conventional usage. See Immunology— A Synthesis (2nd Edition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)). Stereoisomers (e.g., D-amino acids) of the twenty conventional amino acids, unnatural amino acids such as a-, a-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids can also be suitable components for polypeptides of the present invention. Examples of unconventional amino acids include: 4-hydroxyproline, y-carboxyglutamate, £-N, N, N-trimethyllysine, s-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3 -methylhistidine, 5 -hydroxy lysine, o-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxy-terminal direction, in accordance with standard usage and convention.

[0124] Similarly, unless specified otherwise, the left-hand end of single-stranded polynucleotide sequences is the 5' end; the left-hand direction of double-stranded polynucleotide sequences is referred to as the 5' direction. The direction of 5' to 3' addition of nascent RNA transcripts is referred to as the transcription direction; sequence regions on the DNA strand having the same sequence as the RNA and which are 5' to the 5' end of the RNA transcript are referred to as "upstream sequences"; sequence regions on the DNA strand having the same sequence as the RNA and which are 3' to the 3' end of the RNA transcript are referred to as "downstream sequences".

[0125] Conservative amino acid substitutions can encompass non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics and other reversed or inverted forms of amino acid moieties.

[0126] Naturally occurring residues can be divided into classes based on common side chain properties:

[0127] 1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

[0128] 2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;

[0129] 3) acidic: Asp, Glu;

[0130] 4) basic: His, Lys, Arg;

[0131] 5) residues that influence chain orientation: Gly, Pro; and

[0132] 6) aromatic: Trp, Tyr, Phe. For example, non-conservative substitutions can involve the exchange of a member of one of these classes for a member from another class. Such substituted residues can be introduced, for example, into regions of a human antibody that are homologous with non-human antibodies, or into the non-homologous regions of the molecule.

[0133] An "antigen binding protein" as used herein means any protein that binds a specified target antigen. In the instant application, the specified target antigen is the SCGF protein or fragment thereof. " Antigen binding protein" includes but is not limited to antibodies and binding parts thereof, such as immunologically functional fragments. Peptibodies are another example of antigen binding proteins. The term "immunologically functional fragment" (or simply "fragment") of an antibody or immunoglobulin chain (heavy or light chain) antigen binding protein, as used herein, is a species of antigen binding protein comprising a portion (regardless of how that portion is obtained or synthesized) of an antibody that lacks at least some of the amino acids present in a full-length chain but which is still capable of specifically binding to an antigen. Such fragments are biologically active in that they bind to the target antigen and can compete with other antigen binding proteins, including intact antibodies, for binding to a given epitope. In some embodiments, the fragments can block or reduce the likelihood of the interaction between SCGF and its receptor and / or binding partners. In one aspect, such a fragment will retain at least one CDR present in the full-length light or heavy chain, and in some embodiments will comprise a single heavy chain and / or light chain or portion thereof. These biologically active fragments can be produced by recombinant DNA techniques, or can be produced by enzymatic or chemical cleavage of antigen binding proteins, including intact antibodies. Immunologically functional immunoglobulin fragments include, but are not limited to, Fab, a diabody (heavy chain variable domain on the same polypeptide as a light chain variable domain, connected via a short peptide linker that is too short to permit pairing between the two domains on the same chain), Fab', F(ab')2, Fv, domain antibodies and single-chain antibodies, and can be derived from any mammalian source, including but not limited to human, mouse, rat, camelid or rabbit. Further examples of antigen-binding fragments are so-called microantibodies, which are derived from single CDRs. For example, Heap et al., 2005, describe a 17 amino acid residue microantibody derived from the heavy chain CDR3 of an antibody directed against the gpl20 envelope glycoprotein of HIV-1. Other examples include small antibody mimetics comprising two or more CDR regions that are fused to each other, preferably by cognate framework regions. Such a small antibody mimetic comprising VH CDR1 and VL CDR3 linked by the cognate VH FR2 has been described by Qiu et al., 2007. It is further contemplated that a functional portion of the antigen binding proteins disclosed herein, for example, one or more CDRs, could be covalently bound to a second protein or to a small molecule to create a therapeutic agent directed to a particular target in the body, possessing bifunctional therapeutic properties, or having a prolonged serum half-life. As will be appreciated by one of skill in the art, an antigen binding protein can include non-protein components.

[0134] The term "antibody" refers to an intact immunoglobulin of any isotype, or a fragment thereof that can compete with the intact antibody for specific binding to the target antigen, and includes, for instance, chimeric, humanized, fully human, and bispecific antibodies. An "antibody" is a species of an antigen binding protein. An intact antibody will generally comprise at least two full-length heavy chains and two full-length light chains, but in some instances can include fewer chains such as antibodies naturally occurring in camelids which can comprise only heavy chains. Antibodies can be derived solely from a single source, or can be "chimeric," that is, different portions of the antibody can be derived from two different antibodies as described further below. The antigen binding proteins, antibodies, or binding fragments can be produced in hybridomas, by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Unless otherwise indicated, the term "antibody" includes, in addition to antibodies comprising two full-length heavy chains and two full-length light chains, derivatives, variants, fragments, and mutants thereof, examples of which are described below. Furthermore, unless explicitly excluded, antibodies include monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), chimeric antibodies, humanized antibodies, human antibodies, antibody fusions (sometimes referred to herein as "antibody conjugates"), and fragments thereof, respectively. In some embodiments, the term also encompasses peptibodies.

[0135] Naturally occurring antibody structural units typically comprise a tetramer. Each such tetramer typically is composed of two identical pairs of polypeptide chains, each pair having one full-length "light" (in certain embodiments, about 25 kDa) and one full-length "heavy" chain (in certain embodiments, about 50-70 kDa). The amino-terminal portion of each chain typically includes a variable region of about 100 to 110 or more amino acids that typically is responsible for antigen recognition. The carboxy-terminal portion of each chain typically defines a constant region that can be responsible for effector function. Human light chains are typically classified as kappa and lambda light chains. Heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, but not limited to, IgGl, IgG2, IgG3, and IgG4. IgM has subclasses including, but not limited to, IgMl and IgM2. IgA is similarly subdivided into subclasses including, but not limited to, IgAl and IgA2. Within full-length light and heavy chains, typically, the variable and constant regions are joined by a " J" region of about 12 or more amino acids, with the heavy chain also including a " D" region of about 10 more amino acids. See, e.g. Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N. Y. (1989)). The variable regions of each light / heavy chain pair typically form the antigen binding site.

[0136] The variable regions typically exhibit the same general structure of relatively conserved framework regions (FR) joined by three hyper variable regions, also called complementarity determining regions or CDRs. The CDRs from the two chains of each pair typically are aligned by the framework regions, which can enable binding to a specific epitope. From N-terminal to C-terminal, both light and heavy chain variable regions typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is typically in accordance with the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk, J. Mol. Biol., 196:901-917 (1987); Chothia et al., Nature, 342:878-883 (1989).

[0137] In certain embodiments, an antibody heavy chain binds to an antigen in the absence of an antibody light chain. In certain embodiments, an antibody light chain binds to an antigen in the absence of an antibody heavy chain. In certain embodiments, an antibody binding region binds to an antigen in the absence of an antibody light chain. In certain embodiments, an antibody binding region binds to an antigen in the absence of an antibody heavy chain. In certain embodiments, an individual variable region specifically binds to an antigen in the absence of other variable regions. In certain embodiments, definitive delineation of a CDR and identification of residues comprising the binding site of an antibody is accomplished by solving the structure of the antibody and / or solving the structure of the antibody-ligand complex. In certain embodiments, that can be accomplished by any of a variety of techniques known to those skilled in the art, such as X-ray crystallography. In certain embodiments, various methods of analysis can be employed to identify or approximate the CDR regions. Examples of such methods include, but are not limited to, the Kabat definition, the Chothia definition, the AbM definition and the contact definition.

[0138] The Kabat definition is a standard for numbering the residues in an antibody and is typically used to identify CDR regions. See, e.g. Johnson & Wu, Nucleic Acids Res., 28: 214-8 (2000). The Chothia definition is similar to the Kabat definition, but the Chothia definition takes into account positions of certain structural loop regions. See, e.g. Chothia et al., J. Mol. Biol., 196: 901-17 (1986); Chothia et al., Nature, 342: 877-83 (1989). The AbM definition uses an integrated suite of computer programs produced by Oxford Molecular Group that model antibody structure. See, e.g. Martin et al., Proc Natl Acad Sci (USA), 86:9268-9272 (1989); " AbM™, A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK; Oxford Molecular, Ltd. The AbM definition models the tertiary structure of an antibody from primary sequence using a combination of knowledge databases and ab initio methods, such as those described by Samudrala et al., " Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach," in PROTEINS, Structure, Function and Genetics Suppl., 3:194-198 (1999). The contact definition is based on an analysis of the available complex crystal structures. See, e.g. MacCallum et al., J. Mol. Biol., 5:732-45 (1996).

[0139] By convention, the CDR regions in the heavy chain are typically referred to as Hl, H2, and H3 and are numbered sequentially in the direction from the amino terminus to the carboxy terminus. The CDR regions in the light chain are typically referred to as LI, L2, and L3 and are numbered sequentially in the direction from the amino terminus to the carboxy terminus.

[0140] The term "light chain" includes a full-length light chain and fragments thereof having sufficient variable region sequence to confer binding specificity. A full-length light chain includes a variable region domain, VL, and a constant region domain, CL. The variable region domain of the light chain is at the amino-terminus of the polypeptide. Light chains include kappa chains and lambda chains.

[0141] The term "heavy chain" includes a full-length heavy chain and fragments thereof having sufficient variable region sequence to confer binding specificity. A full-length heavy chain includes a variable region domain, VH, and three constant region domains, CHI, CH2, and CH3. The VH domain is at the amino-terminus of the polypeptide, and the CH domains are at the carboxylterminus, with the CH3 being closest to the carboxy-terminus of the polypeptide. Heavy chains can be of any isotype, including IgG (including IgGl, IgG2, IgG3 and IgG4 subtypes), IgA (including IgAl and IgA2 subtypes), IgM and IgE.

[0142] A bispecific or bifunctional antibody typically is an artificial hybrid antibody having two different heavy / light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods including, but not limited to, fusion of hybridomas or linking of Fab' fragments. See, e.g. Songsivilai et al., Clin. Exp. Immunol., 79: 315-321 (1990); Kostelny et al., J. Immunol., 148:1547-1553 (1992).

[0143] Some species of mammals also produce antibodies having only a single heavy chain.

[0144] Each individual immunoglobulin chain is typically composed of several "immunoglobulin domains," each consisting of roughly 90 to 110 amino acids and having a characteristic folding pattern. These domains are the basic units of which antibody polypeptides are composed. In humans, the IgA and IgD isotypes contain four heavy chains and four light chains; the IgG and IgE isotypes contain two heavy chains and two light chains; and the IgM isotype contains five heavy chains and five light chains. The heavy chain C region typically comprises one or more domains that can be responsible for effector function. The number of heavy chain constant region domains will depend on the isotype. IgG heavy chains, for example, contain three C region domains known as CHI, CH2 and CH3. The antibodies that are provided can have any of these isotypes and subtypes. In certain embodiments of the present invention, an anti-SCGF antibody is of the IgG2 or IgG4 subtype.

[0145] The term "variable region" or "variable domain" refers to a portion of the light and / or heavy chains of an antibody, typically including approximately the amino-terminal 120 to 130 amino acids in the heavy chain and about 100 to 110 amino terminal amino acids in the light chain. In certain embodiments, variable regions of different antibodies differ extensively in amino acid sequence even among antibodies of the same species. The variable region of an antibody typically determines specificity of a particular antibody for its target.

[0146] The term "compete" when used in the context of antigen binding proteins (e.g. neutralizing antigen binding proteins or neutralizing antibodies) that compete for the same epitope means competition between antigen binding proteins as determined by an assay in which the antigen binding protein (e.g. antibody or immunologically functional fragment thereof) being tested prevents or inhibits (e.g. reduces) specific binding of a reference antigen binding protein (e.g. a ligand, or a reference antibody) to a common antigen (e.g. SCGF or a fragment thereof). Numerous types of competitive binding assays can be used to determine if one antigen binding protein competes with another, for example: solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see e.g. Stahli et al., 1983, Methods in Enzymology 9:242-253); solid phase direct biotin-avidin EIA (see e.g. Kirkland et al., 1986, J. Immunol. 137:3614-3619) solid phase direct labeled assay, solid phase direct labeled sandwich assay (see e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press ); solid phase direct label RIA using 1-125 label (see, e.g. Morel et al., 1988, Molec. Immunol. 25:7-15); solid phase direct biotin-avidin EIA (see, e.g. Cheung, et al., 1990, Virology 176:546-552); and direct labelled RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82). Typically, such an assay involves the use of purified antigen bound to a solid surface or cells bearing either of these, an unlabelled test antigen binding protein and a labelled reference antigen binding protein. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test antigen binding protein. Usually the test antigen binding protein is present in excess. Antigen binding proteins identified by competition assay (competing antigen binding proteins) include antigen binding proteins binding to the same epitope as the reference antigen binding proteins and antigen binding proteins binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference antigen binding protein for steric hindrance to occur. Additional details regarding methods for determining competitive binding are provided in the examples herein. Usually, when a competing antigen binding protein is present in excess, it will inhibit (e.g., reduce) specific binding of a reference antigen binding protein to a common antigen by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75% or 75% or more. In some instances, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

[0147] The term "antigen" refers to a molecule or a portion of a molecule capable of being bound by a selective binding agent, such as an antigen binding protein (including e.g. an antibody or immunological functional fragment thereof). In some embodiments, the antigen is capable of being used in an animal to produce antibodies capable of binding to that antigen. An antigen can possess one or more epitopes that are capable of interacting with different antigen binding proteins e.g. antibodies.

[0148] The term "epitope" includes any determinant capable being bound by an antigen binding protein, such as an antibody or to a T-cell receptor. An epitope is a region of an antigen that is bound by an antigen binding protein that targets that antigen, and when the antigen is a protein, includes specific amino acids that directly contact the antigen binding protein. Most often, epitopes reside on proteins, but in some instances can reside on other kinds of molecules, such as nucleic acids. Epitope determinants can include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and can have specific three dimensional structural characteristics, and / or specific charge characteristics. Generally, antibodies specific for a particular target antigen will preferentially recognize an epitope on the target antigen in a complex mixture of proteins and / or macromolecules.

[0149] As used herein, the term "antibody-like protein" refers to a protein that has been engineered (e.g. by mutagenesis of loops) to specifically bind to a target molecule. Typically, such an antibodylike protein comprises at least one variable peptide loop attached at both ends to a protein scaffold. This double structural constraint greatly increases the binding affinity of the antibody-like protein to levels comparable to that of an antibody. The length of the variable peptide loop typically consists of 10 to 20 amino acids. The scaffold protein may be any protein having good solubility properties. Preferably, the scaffold protein is a small globular protein. Antibody-like proteins include affibodies, anticalins, and designed ankyrin repeat proteins (for review see: Binz H. K. et al. (2005) Engineering novel binding proteins from nonimmunoglobulin domains. Nat. Biotechnol. 23(10): 1257-1268). Further examples of antibody like proteins are lipoprotein-associated coagulation inhibitor (LACI-D1); affilin, e.g. human-γ B crystalline or human ubiquitin; cystatin; Sac7D from Sulfolobus acidocaldarius; lipocalin; SH3 domain of Fyn; Kunits domain of a protease inhibitor; monobodies, e.g. the 10th type III domain of fibronectin; adnectins: cysteine knot miniproteins knottins; atrimers; evibodies, e.g., affibodies, e.g. three-helix bundle from Z-domain of protein A from Staphylococcus aureus; Trans-bodies, e.g. human transferrin; tetranectins, e.g. monomeric or trimeric human C-type lectin domain; microbodies, e.g. trypsin-inhibitor-II; an armadillo repeat protein; tetraspanins, B-type lection domain containing elements, EZH type domain containing elements, avimers (Weidle UH, et al., (2013), Cancer Genomics Proteomics; 10(4): 155-68), nanofitins (Mouratou B, Behar G, Paillard-Laurance L, Colinet S, Pecorari F., (2012) Methods Mol Biol.; 805:315-31) and affilins (Weidle UH, et al., (2013), Cancer Genomics Proteomics; 10(4): 155-68). Antibody-like proteins can be derived from large libraries of mutants, e.g. be panned from large phage display libraries and can be isolated in analogy to regular antibodies. Also, antibody-like binding proteins can be obtained by combinatorial mutagenesis of surface-exposed residues in globular proteins. Antibody-like proteins are sometimes referred to as "peptide aptamers".

[0150] Certain antigen binding proteins described herein are antibodies or are derived from antibodies. In certain embodiments, the polypeptide structure of the antigen binding proteins is based on antibodies, including, but not limited to, monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), chimeric antibodies, humanized antibodies, human antibodies, antibody fusions (sometimes referred to herein as "antibody conjugates"), and fragments thereof, respectively.

[0151] An " Fc" region comprises two heavy chain fragments comprising the CHI and CH2 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains.

[0152] A " Fab fragment" comprises one light chain and the CHI and variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.

[0153] A " Fab1fragment" comprises one light chain and a portion of one heavy chain that contains the VH domain and the CHI domain and also the region between the CHI and CH2 domains, such that an interchain disulfide bond can be formed between the two heavy chains of two Fab' fragments to form an F(ab')2molecule.

[0154] A " F(ab')2fragment" contains two light chains and two heavy chains containing a portion of the constant region between the CHI and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains. A F(ab')2fragment thus is composed of two Fab' fragments that are held together by a disulfide bond between the two heavy chains.

[0155] The " Fv region" comprises the variable regions from both the heavy and light chains, but lacks the constant regions.

[0156] " Single-chain antibodies" are Fv molecules in which the heavy and light chain variable regions have been connected by a flexible linker to form a single polypeptide chain, which forms an antigen binding region. Single chain antibodies are discussed in detail in International Patent Application Publication No. WO 88 / 01649 and United States Patent Nos. 4,946,778 and No.

[0157] 5,260,203.

[0158] A "domain antibody" is an immunologically functional immunoglobulin fragment containing only the variable region of a heavy chain or the variable region of a light chain. In some instances, two or more VHregions are covalently joined with a peptide linker to create a bivalent domain antibody. The two VHregions of a bivalent domain antibody can target the same or different antigens.

[0159] A "bivalent antigen binding protein" or "bivalent antibody" comprises two antigen binding sites. In some instances, the two binding sites have the same antigen specificities. Bivalent antigen binding proteins and bivalent antibodies can be bispecific see infra. A bivalent antibody other than a "multispecific" or "multifunctional" antibody, in certain embodiments, typically is understood to have each of its binding sites identical. A "multispecific antigen binding protein" or "multispecific antibody" is one that targets more than one antigen or epitope.

[0160] A "bispecific," "dual-specific" or "bifunctional" antigen binding protein or antibody is a hybrid antigen binding protein or antibody, respectively, having two different antigen binding sites. Bispecific antigen binding proteins and antibodies are a species of multispecific antigen binding protein antibody and can be produced by a variety of methods including, but not limited to, fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai and Lachmann, 1990, Clin. Exp. Immunol. 79:315-321; Kostelny etal., 1992, J. Immunol. 148:1547-1553. The two binding sites of a bispecific antigen binding protein or antibody will bind to two different epitopes, which can reside on the same or different protein targets.

[0161] An antigen binding protein is said to "specifically bind" its target antigen when the dissociation constant (Kd) is <10<-7> M. The antigen binding protein specifically binds antigen with "high affinity" when the Kd is <5 x 10'9M, and with "very high affinity" when the Kd is <5x 10"10M. In one embodiment, the antigen binding protein has a Kd of <10'9M. In one embodiment, the off-rate is < 1 x IO'5. In other embodiments, the antigen binding proteins will bind to human SCGF with a Kd of between about 10'9M and 10'13M, and in yet another embodiment the antigen binding protein will bind with a Kd <5 x 10'10M. As will be appreciated by one of skill in the art, in some embodiments, any or all of the antigen binding fragments can specifically bind to SCGF.

[0162] An antigen binding protein is "selective" when it binds to one target more tightly than it binds to a second target.

[0163] " Antigen binding region" means a protein, or a portion of a protein, that specifically binds a specified antigen (e.g., a paratope). For example, that portion of an antigen binding protein that contains the amino acid residues that interact with an antigen and confer on the antigen binding protein its specificity and affinity for the antigen is referred to as "antigen binding region." An antigen binding region typically includes one or more "complementary binding regions" (" CDRs"). Certain antigen binding regions also include one or more "framework" regions. A " CDR" is an amino acid sequence that contributes to antigen binding specificity and affinity. " Framework" regions can aid in maintaining the proper conformation of the CDRs to promote binding between the antigen binding region and an antigen. Structurally, framework regions can be located in antibodies between CDRs.

[0164] In certain aspects, recombinant antigen binding proteins that bind SCGF, for example human SCGF, are provided. In this context, a "recombinant antigen binding protein" is a protein made using recombinant techniques, i.e. through the expression of a recombinant nucleic acid as described herein. Methods and techniques for the production of recombinant proteins are well known in the art.

[0165] The term "treat" and "treatment" includes therapeutic treatments, prophylactic treatments, and applications in which one reduces the risk that a subject will develop a disorder or other risk factor. Treatment does not require the complete curing of a disorder and encompasses embodiments in which one reduces symptoms or underlying risk factors.

[0166] The term "prevent" does not require the 100% elimination of the possibility of an event. Rather, it denotes that the likelihood of the occurrence of the event has been reduced in the presence of the compound or method.

[0167] Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques can be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures can be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (1989)). Unless specific definitions are provided, the nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques can be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0168] The term "chimeric antibody" refers to those antibodies wherein one portion of each of the amino acid sequences of heavy and light chains is homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular class, while the remaining segment of the chain is homologous to corresponding sequences in another species or class. Typically the variable region of both light and heavy chains mimics the variable regions of antibodies derived from one species of mammals, while the constant portions are homologous to sequences of antibodies derived from another. One clear advantage to such chimeric forms is that the variable region can conveniently be derived from presently known sources using readily available B-cells or hybridomas from non-human host organisms in combination with constant regions derived from, for example, human cell preparations. While the variable region has the advantage of ease of preparation and the specificity is not affected by the source, the constant region being human is less likely to elicit an immune response from a human subject when the antibodies are injected than would the constant region from a non-human source. However, the definition is not limited to this particular example.

[0169] The term "humanized antibody" refers to a molecule having an antigen binding site that is substantially derived from an immunoglobulin from a non-human species, wherein the remaining immunoglobulin structure of the molecule is based upon the structure and / or sequence of a human immunoglobulin. The antigen binding site may either comprise complete variable domains fused onto constant domains or only the complementarity determining regions (CDR) grafted onto appropriate framework regions in the variable domains. Antigen-binding sites may be wild-type or modified by one or more amino acid substitutions, e.g. modified to resemble human immunoglobulins more closely. Some forms of humanized antibodies preserve all CDR sequences (for example a humanized mouse antibody which contains all six CDRs from the mouse antibody). Other forms have one or more CDRs which are altered with respect to the original antibody.

[0170] Different methods for humanizing antibodies are known to the skilled person, as reviewed by Almagro & Fransson, 2008. The review article by Almagro & Fransson is briefly summarized in the following. Almagro & Fransson distinguish between rational approaches and empirical approaches. Rational approaches are characterized by generating few variants of the engineered antibody and assessing their binding or any other property of interest. If the designed variants do not produce the expected results, a new cycle of design and binding assessment is initiated. Rational approaches include CDR grafting, Resurfacing, Superhumanization, and Human String Content Optimization. In contrast, empirical approaches are based on the generation of large libraries of humanized variants and selection of the best clones using enrichment technologies or high-throughput screening. Accordingly, empirical approaches are dependent on a reliable selection and / or screening system that is able to search through a vast space of antibody variants. In vitro display technologies, such as phage display and ribosome display fulfill these requirements and are well-known to the skilled person. Empirical approaches include FR libraries, Guided selection, Framework-shuffling, and Humaneering.

[0171] CDR grafting:

[0172] A CDR grafting protocol typically comprises three decision-making points: (1) definition of regions determining the specificity of the donor antibody, i.e. the target for grafting, (2) identification of a source of human sequences to be utilized as FR donors, and (3) selection of residues outside of the region defining the specificity, i.e. determining amino acid positions that are targets for back mutation to restore or improve the affinity of the humanized antibody.

[0173] (1) Regions determining the antibody specificity

[0174] The experimental structure of the non-human antibody in complex with the antigen provides a detailed map of residues in contact with the antigen and therefore those responsible for determining its specificity. The structural information can be complemented with alanine scanning mutagenesis and / or combinatorial mutagenesis to identify the residues contributing most to the binding energy or to the functional paratope. Since the functional paratope is a subset of the residues in contact, grafting only the functional paratope would reduce the number of non-human residues in the humanized product. However, only in rare cases are the experimental structure of the antigenantibody complex and / or the functional paratope available at the beginning of a humanization protocol. In absence of a precise definition of residues responsible for a given antibody specificity, CDRs are often employed as regions defining the specificity. It is also possible to use a combination of CDR and HV loop as targets for grafting. To reduce the number of residues to be grafted on the human FRs, SDR grafting has been described, i.e. the grafting of specificity-determining residues (SDRs).

[0175] (2) Source of human FRs

[0176] The second step in a typical CDR grafting protocol is to identify human FR donors. Initial works utilized FRs of human antibodies of known structure, regardless of their homology to the non-human antibody. This approach is known as "fixed FR method". Later works used human sequences having the highest homology to the non-human antibody. This approach has been termed " Best Fit". While "best fit" strategies tend to result in antibodies with higher affinity, other parameters such as low immunogenicity and production yields have to be taken into account, too, when choosing an FR for humanization. Thus, combinations of "best fit" and "fixed FR" are also possible. For example, the VL part can be humanized according to the fixed FR method and the VH part can be humanized according to the best fit method, or vice versa.

[0177] Two sources of human sequences have been utilized: mature and germline sequences. Mature sequences, which are products of immune responses, carry somatic mutations generated by random processes and are not under the species selection, resulting in potential immunogenic residues. Thus, to avoid immunogenic residues, human germline genes have increasingly been utilized as source of FR donors. Nucleotide sequences of human germline FRs are disclosed e.g. in Appendices A and B of the article by Dall'Acqua et al, 2005. Furthermore, germline gene based antibodies tend to be more flexible as compared to mature antibodies. This higher flexibility is thought to better accommodate diverse CDRs with fewer or no back mutations into the FR to restore the affinity of the humanized antibody.

[0178] (3) Back mutations to restore or enhance affinity

[0179] Commonly, affinity decreases after CDR grafting as a consequence of incompatibilities between non-human CDRs and human FRs. Therefore, the third step in a typical CDR grafting protocol is to define mutations that would restore or prevent affinity losses. Back mutations have to be carefully designed based on the structure or a model of the humanized antibody and tested experimentally. A web site for automated antibody modelling called WAM can be found at the URL http: / / antibody.bath.ac.uk. Software for protein structure modelling can be downloaded at the sites http: / / salilab.org / modeller / modeller.html (Modeller) and http: / / spdbv.vital-it.ch (Swiss PdbViewer).

[0180] Resurfacing

[0181] Resurfacing is similar to CDR grafting and shares the first two decision-making points. In contrast to CDR grafting, resurfacing retains the non-exposed residues of the non-human antibody. Only surface residues in the non-human antibody are changed to human residues.

[0182]

[0183] While CDR grafting relies on the FR comparison between the non-human and the human sequences, superhumanization is based on a CDR comparison so that FR homology is irrelevant. The approach includes a comparison of the non-human sequence with the functional human germline gene repertoire. Those genes encoding the same or closely related canonical structures to the murine sequences are then selected. Next, within the genes sharing the canonical structures with the non-human antibody, those with highest homology within the CDRs are chosen as FR donors. Finally, the non-human CDRs are grafted onto these FRs.

[0184] Human String Content Optimization

[0185] This approach is based on a metric of antibody "humanness", termed Human String Content (HSC). In short, this approach compares the mouse sequence with the repertoire of human germline genes. Differences are scored as HSC. The target sequence is then humanized by maximizing its HSC rather than using a global identity measure to generate multiple diverse humanized variants. Framework libraries

[0186]

[0187] : FRh

[0188] In the FR library approach, a collection of residue variants are introduced at specific positions in the FR followed by panning of the library to select the FR that best supports the grafted CDR. Thus, this approach resembles CDR grafting but instead of creating a few back mutations in the FR, a combinatorial library of typically more than 100 mutational variants is constructed.

[0189] Guided Selection

[0190] This approach includes combining the VH or VL domain of a given non-human antibody specific for a particular antigen with a human VH and VL library. Subsequently, specific human V domains are selected against the antigen of interest. For example, a non-human antibody can be humanized by first combining the non-human VH with a library of human light chains. The library is then selected against the target antigen by phage display and the selected VL is cloned into a library of human VH chains and selected against the target antigen. It is also possible to start with combining the non-human VL with a library of human heavy chains. The library is then selected against the target antigen by phage display and the selected VH is cloned into a library of human VL chains and selected against the target antigen. As a result, a fully human antibody with similar affinity as the non-human antibody can be isolated. To avoid the occurrence of an epitope drift, it is possible to implement a so-called inhibition ELISA, which allows for the selection of clones recognizing the same epitope as the parent antibody. Alternatively, CDR retention can be applied to avoid an epitope drift. In CDR retention, one or more non-human CDRs are retained, preferably the heavy chain CDR3, since this CDR is at the center of the antigen binding site.

[0191] Framework shuffling (abbreviated: FR shuffling)

[0192] In the FR shuffling approach, whole FRs are combined with the non-human CDRs. Using FR shuffling, Dall' Acqua and co-workers humanized a murine antibody. All six CDRs of the murine antibody were cloned into a library containing all human germline gene FRs (Dall1Acqua et al., 2005). The libraries were screened for binding in a two-step selection process, first humanizing VL, followed by VH. In a later study, a one-step FR shuffling process was successfully used (Damschroder et al., 2007). Oligonucleotide sequences encoding all known human germline light chain (K) frameworks are disclosed in Dall' Acqua et al., 2005, as Appendix A. Oligonucleotide sequences encoding all known human germline heavy chain frameworks are disclosed in Dall'Acqua et al., 2005, as Appendix B.

[0193] Humaneering

[0194] Humaneering allows for isolation of antibodies that are 91-96% homologous to human germline gene antibodies. The method is based on experimental identification of essential minimum specificity determinants (MSDs) and on sequential replacement of non-human fragments into libraries of human FRs and assessment of binding. It begins with regions of the CDR3 of non-human VH and VL chains and progressively replaces other regions of the non-human antibody into the human FRs, including the CDR1 and CDR2 of both VH and VL.

[0195] The methods for humanizing antibodies explained above are preferred when generating humanized antibodies that specifically bind to the conformational epitopes described herein. Nevertheless, the present invention is not limited to the above-mentioned methods for humanizing antibodies.

[0196] Some of the aforementioned humanization methods can be performed without information about the FR sequences in the donor antibody, namely the " Fixed FR Method" (a variant of CDR-grafting), Superhumanization, Framework-shuffling, and Humaneering. Variations of the "fixed FR method" were successfully carried out by Qin et al., 2007 and Chang et al., 2007. In particular, Qin et al. constructed an antibody fragment comprising a human heavy chain variable region in which the three CDR regions were replaced by antigenic peptides, which were derived from the CDR sequences of a murine antibody. Chang et al. continued these experiments and constructed a scFv fragment, in which all CDRs from the VH part and CDR3 from the VL part were replaced by antigenic peptides, which were derived from the CDR sequences of a murine antibody.

[0197] As used herein, "human antibodies" include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). Human antibodies of the invention include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulin and that do not express endogenous immunoglobulins, as described for example in U. S. Patent No. 5,939,598 by Kucherlapati & Jakobovits. Embodiments

[0198] In the following passages different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0199] In the work leading to the present invention, it was surprisingly shown that SCGF is the main driver of stem cell properties in cancer stem cells that are causative for developing therapy resistance. Intervention on the level of SCGF using the antigen binding protein of the present invention abrogates the deteriorating induction of this cancer stem cell-like properties.

[0200] Based on these results the present invention provides in a first aspect an antigen binding protein that specifically binds to Stem Cell Growth Factor (SCGF). The antigen binding protein competes for binding to SCGF with a reference antibody comprising a heavy chain variable region of the amino acid sequence in SEQ ID NO: 1; and a light chain variable region of the amino acid sequence in SEQ ID NO: 2.

[0201] The reference antibody of the first aspect of the invention comprises a heavy chain variable region with the following amino acid sequence (SEQ ID NO: 1):

[0202] EVQLQQSGPELVKPGASVKISCKASGYSFTGYYMHWVKQGPGKSLEWIGLI I PHNGGSRNNQRFKGK ATLTVDKSSSTAYMELRSLTSEDSAVYYCARQVYYYDGSYVAWFANWGQGTLVTVSA

[0203] The reference antibody further comprises a light chain variable region with the following amino acid sequence (SEQ ID NO: 2):

[0204] DIVMTQSQKFMSTAVGDRVSITCKASQNVGTAVAWYQQKPGQSPKLLIYSASNRFTGVPDRFTGSGS GTDFTLTISNMQSEDLADYFCQQYSSYPLTFGAGTKLELK

[0205] In a second aspect of the invention an antigen binding protein that specifically binds to SCGF, wherein said antigen binding protein comprises either

[0206] (i) a combination of a light chain variable domain and a heavy chain variable domain selected from the group of combinations consisting of:

[0207] a heavy chain variable domain having a sequence with at least 90% identity to SEQ ID NO: 1; and

[0208] a light chain variable domain having a sequence with at least 90% identity to SEQ ID NO: 2; or

[0209] (ii) a complementarity determining region 3 of the heavy chain (CDRH3) comprising or consisting of the amino acid sequence of SEQ ID NO: 5 and a complementarity determining region 3 of the light chain (CDRL3) comprising or consisting of the amino acid sequence of SEQ ID NO: 8.

[0210] The complementarity determining region 3 of the heavy chain (CDRH3) of SEQ ID NO: 5 has the following amino acid sequence: QVYYYDGSYVAWFAN

[0211] The complementarity determining region 3 of the light chain (CDRL3) of SEQ ID NO: 8 has the following amino acid sequence: QQYSSYPLT

[0212] One of the purposes of the antigen binding protein of the present invention is to target the functional role of SCGF as a main driver of inducing stem cell properties resulting in chemotherapy resistance, production of extracellular matrix, protection from immune cell recognition. The antigen binding protein abrogates the deteriorating induction of this cancer stem cell-like properties

[0213] In a preferred embodiment of the first and second aspect of the invention the antigen binding protein comprises an antibody, a SCGF-binding fragment of an antibody or an antibody-like protein.

[0214] In a preferred embodiment of the first and second aspect of the invention the antigen binding protein, wherein:

[0215] (i) the antibody is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, or a rodent, in particular a mouse antibody or from any other species; and / or

[0216] (ii) the antibody is a mono-specific, bi-specific, tri-specific, or multi-specific antibody; and / or (ii) the antibody is a single chain antibody, a single chain variable fragment antibody, a diabody, a Fab fragment, or an F(ab)2 fragment; and / or

[0217] (iv) the antibody is selected from an IgA, IgE, IgG, in particular IgGl, an IgG2 antibody, an IgG3 antibody, and an IgG4 antibody; and IgM; or

[0218] (v) the antibody-like protein is selected from the group consisting of lipoprotein-associated coagulation inhibitor (LACI-D1); affilin, e.g. human-γ B crystalline or human ubiquitin; cystatin; Sac7D from Sulfolobus acidocaldarius; lipocalin, anticalin; designed ankyrin repeat domains (DARPins); SH3 domain of Fyn; Kunits domain of a protease inhibitor; monobodies, e.g. the 10thtype III domain of fibronectin; adnectins: cysteine knot miniproteins knottins; atrimers; evibodies, e.g., affibodies, e.g. three-helix bundle from Z- domain of protein A from Staphylococcus aureus Trans-bodies, e.g. human transferrin; tetranectins, e.g. monomeric or trimeric human C-type lectin domain; microbodies, e.g. trypsin-inhibitor-II; an armadillo repeat protein; tetraspanins, B-type lection domain containing elements, EZH type domain containing elements, avimers, nanofitins and affilins. In a preferred embodiment of the first and second aspect of the invention the antigen binding protein comprises an antibody that is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, or a rodent from any other species. Preferably the rodent antibody is a mouse antibody.

[0219] In a preferred embodiment of the first and second aspect of the invention the antigen binding protein comprises an antibody that is selected from the group consisting of a human antibody and a humanized antibody. Methods how to obtain such human and humanized antibody are provided above.

[0220] In a preferred embodiment of the first and second aspect of the invention the antigen binding protein comprises an antibody that is a mono-specific, bi-specific, tri-specific, or multi-specific antibody.

[0221] In a preferred embodiment of the first and second aspect of the invention the antigen binding protein comprises an antibody that is a single chain antibody, a single chain variable fragment antibody, a diabody, a Fab fragment, or an F(ab)2 fragment.

[0222] In a preferred embodiment of the first and second aspect of the invention the antigen binding protein comprises an antibody that is an IgA, IgE, IgG and IgM

[0223] In a preferred embodiment of the first and second aspect of the invention, wherein the antibody is an IgG antibody, the antibody is selected from the group comprising an IgGl, an IgG2, an IgG3, and an IgG4 antibody.

[0224] In a preferred embodiment of the first and second aspect of the invention the antibody-like protein is selected from the group consisting of lipoprotein-associated coagulation inhibitor (LACI-Dl); affilin, e.g. human-γ B crystalline or human ubiquitin; cystatin; Sac7D from Sulfolobus acidocaldarius,' lipocalin, anticalin; designed ankyrin repeat domains (DARPins); SH3 domain of Fyn; Kunits domain of a protease inhibitor; monobodies, e.g. the 10thtype III domain of fibronectin; adnectins: cysteine knot miniproteins knottins; atrimers; evibodies, e.g., affibodies, e.g. three-helix bundle from Z-domain of protein A from Staphylococcus aureus, Trans-bodies, e.g. human transferrin; tetranectins, e.g. monomeric or trimeric human C-type lectin domain; microbodies, e.g. trypsin-inhibitor-II; an armadillo repeat protein; tetraspanins, B-type lection domain containing elements, EZH type domain containing elements, avimers, nanofitins and affilins.

[0225] In a preferred embodiment of the first and second aspect of the invention the antigen binding protein has at least one of the following characteristics:

[0226] a. binds to SCGF with a Kdof less than 1 nM, of less than 100 pM, of less than 10 pM, or of less than 5 pM; and / or b. blocks the binding of SCGF to a cell responsive to SCGF with an IC50of less than 1 nM, preferably less than 200 pM; and / or

[0227] c. decreases secretion of pro-angiogenic cytokines, sternness-related cytokines, immunosuppressive molecules and / or inflammatory cytokines and / or chemokines. In a preferred embodiment of the first and second aspect of the invention the antigen binding protein binds to SCGF with a Ka of less than 1 nM, of less than 100 pM, of less than 10 pM, or of less than 5 pM.

[0228] In a preferred embodiment of the first and second aspect of the invention the antigen binding protein blocks the binding of SCGF to a cell responsive to SCGF with an IC50 of less than 1 nM, preferably less than 200 pM

[0229] In a preferred embodiment of the first and second aspect of the invention the antigen binding protein decreases secretion of pro-angiogenic cytokines, sternness-related cytokines, immunosuppressive molecules and / or inflammatory cytokines and / or chemokines.

[0230] In a preferred embodiment of the first and second aspect of the invention the antigen binding protein is further comprising at least one:

[0231] (i) antigen binding protein specifically binding to a target different from SCGF, in particular to the ectodomain of a protein; and / or

[0232] (ii) a toxin, and / or

[0233] (iii) a label, in particular a radionuclide; and / or

[0234] (iv) a polypeptide comprising or consisting of a transmembrane domain and, optionally an endo domain.

[0235] Examples of alternative (iv) would include chimeric antigen receptors (CARs). The term “chimeric antigen receptors” is used in its meaning known in the art of immunology and refers to the combination of the target specificity of an antigen binding protein, such as an antibody, a SCGF-binding fragment of an antibody (including an scFv) or an antibody-like protein, with an immune cell. Such a chimeric construct usually comprises the target binding ecto-domain of an antigen binding domain with a transmembrane- and an endo-domain of an immune receptor such as a T-cell receptor. Well known example of a CAR are CAR-T-cells comprising an scFv and the transmembrane domain and the intracellular domain of a T-cell receptor grafted onto T-cells.

[0236] In a preferred embodiment of the first aspect of the invention, wherein:

[0237] (i) the target different from SCGF is selected from the group consisting of C-Lectin-Type domains, immunomodulatory or homeostasis-regulating domains different from the respective domains of SCGF; and / or (ii) the transmembrane is the transmembrane domain of CD3-zeta or CD28.

[0238] In a preferred embodiment of the first and second aspect of the invention the antigen binding protein is further comprising an antigen binding protein specifically binding to a target different from SCGF selected from the group consisting of C-Lectin-Type domains, immunomodulatory or homeostasis-regulating domains different from the respective domains of SCGF.

[0239] In a preferred embodiment of the first and second aspect of the invention the antigen binding protein is further comprising a polypeptide comprising or consisting of a transmembrane domain and, optionally an endo domain, wherein the transmembrane is the transmembrane domain of CD3-zeta or CD28.

[0240] In a preferred embodiment of the first and second aspect of the invention, wherein the other or similar C-Lectin-Type domains or immunomodulatory or homeostasis-regulating domains are selected from the group consisting of Stem Cell Factor (SCF), GM-CSF, interleukin-6, PD-1, PD-Ll, PD-L2, 0X40, CD40, CD40L, LAG3, TIM-3, CTLA-4 and fibroblast activating protein 1 (FAP alpha).

[0241] In a preferred embodiment of the first and second aspect of the invention the target different from SCGF includes binding to proteins from the group consisting of Dectin- 1 and / or Dectin-2 or the group of mannose receptors (i.e. able to bind branched sugars with terminal mannose, fucose or N-acetyl-glucosamine).

[0242] In a preferred embodiment of the second aspect of the invention the antigen binding protein is further comprising one or more CDRs selected from the group consisting of a CDRH1 comprising or consisting of the amino acid sequence of SEQ ID NO: 3, a CDRH2 comprising or consisting of the amino acid sequence of SEQ ID NO: 4, CDRL1 comprising or consisting of the amino acid sequence of SEQ ID NO: 6 and a CDRL2 comprising or consisting of the amino acid sequence of SEQ ID NO: 7.

[0243] SEQ ID NO: 3 (CDRH1 ): GYYMH

[0244] SEQ ID NO: 4 (CDRH2 ): LIIPHNGGSRNNQRFKG

[0245] SEQ ID NO: 6 (CDRL1 ): KASQNVGTAVA

[0246] SEQ ID NO: 7 (CDRL2 ): SASNRFT

[0247] In a third aspect of the invention an antigen binding protein that specifically binds to Stem Cell Growth Factor (SCGF) comprises a CDRH1 comprising or consisting of the amino acid sequence of SEQ ID NO: 3, a CDRH2 comprising or consisting of the amino acid sequence of SEQ ID NO: 4, a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 5, a CDRL1 comprising or consisting of the amino acid sequence of SEQ ID NO: 6, a CDRL2 comprising or consisting of the amino acid sequence of SEQ ID NO: 7, and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 8; and / or binds to an epitope of SCGF that comprises or consists of the amino acid sequence PVWLGVHD (SEQ ID NO: 11) of SEQ ID NO: 9 is provided.

[0248] The epitope mapping results are illustrated in Figure 5.

[0249] The human Stem Cell Growth Factor ( / CLEC11 A) amino acid sequence of SEQ ID NO: 9 is MQAAWLLGALWPQLLGFGHGARGAEREWEGGWGGAQEEEREREALMLKHLQEALGLP AGRGDENPAGTVEGKEDWEMEEDQGEEEEEEATPTPS S GPSPSPTPEDIVTYILGRLAGLD A GLHQLHVRLHALDTRWELTQGLRQLRNAAGDTRDAVQALQEAQGRAEREHGRLEGCLK GLRLGHKCFLLSRDFEAQAAAQARCTARGGSLAQPADRQQMEALTRYLRAALAPYNWPV WLGVHDRRAEGLYLFENGORVSFFAWHRSPRPELGAOPSASPHPLSPDOPNGGTLENCVA QASDDGSWWDHD CQRRLYYVCE FPF

[0250] and the encoding nucleic acid sequence of SEP ID NO: 10 is

[0251] 1 agagacgagg agaggaacag gaagagagaa gctgggagaa tcgggaacct gggggctagt

[0252] 61 gacctgcaca cagggcaggg gcactcggca gttcccagag gccacccctc ccaccccaga

[0253] 121 catccagaca tctggaactt tgggtgccaa gagtccagct taatgcaggc agcctggctt

[0254] 181 ttgggggctt tggtggtccc ccagctcttg ggctttggcc atggggctcg gggagcagag

[0255] 241 agggagtggg agggaggctg gggaggtgcc caggaggagg agcgggagag ggaggccctg

[0256] 301 atgctgaagc atctgcagga agccctagga ctgcctgctg ggagggggga tgagaatcct

[0257] 361 gccggaactg ttgagggaaa agaggactgg gagatggagg aggaccaggg ggaggaagag

[0258] 421 gaggaggaag caacgccaac cccatcctcc ggccccagcc cctctcccac ccctgaggac

[0259] 481 atcgtcactt acatcctggg ccgcctggcc ggcctggacg caggcctgca ccagctgcac

[0260] 541 gtccgtctgc acgcgttgga cacccgcgtg gtcgagctga cccaggggct gcggcagctg

[0261] 601 cggaacgcgg caggcgacac ccgcgatgcc gtgcaagccc tgcaggaggc gcagggtcgc

[0262] 661 gccgagcgcg agcacggccg cttggagggc tgcctgaagg ggctgcgcct gggccacaag

[0263] 721 tgcttcctgc tctcgcgcga cttcgaagct caggcggcgg cgcaggcgcg gtgcacggcg

[0264] 781 cggggcggga gcctggcgca gccggcagac cgccagcaga tggaggcgct cactcggtac

[0265] 841 ctgcgcgcgg cgctcgctcc ctacaactgg cccgtgtggc tgggcgtgca cgatcggcgc

[0266] 901 gccgagggcc tctacctctt cgaaaacggc cagcgcgtgt ccttcttcgc ctggcatcgc

[0267] 961 tcaccccgcc ccgagctcgg cgcccagccc agcgcctcgc cgcatccgct cagcccggac

[0268] 1021 cagcccaacg gtggcacgct cgagaactgc gtggcgcagg cctctgacga cggctcctgg

[0269] 1081 tgggaccacg actgccagcg gcgtctctac tacgtctgcg agttcccctt ctagcggggc

[0270] 1141 cggtaccccg cctccctgcc catcccacca cccggccttt ccctgcgccg tgcccaccct

[0271] 1201 cctccggaat ctcccttccc ttcctggcca cgaatggcag cgtcctcccc gacccccagt

[0272] 1261 ctgggcgctt ctgggagggc tcttgcggtg ccggcactcc tccttgttag tgtctttcct

[0273] 1321 tgaaggggcg ggcaccaggc taggtccggt gccaataaat ccttgtggaa tctga^

[0274] In a preferred embodiment of the third aspect of the invention the antigen binding protein comprises a heavy chain variable domain having a sequence with at least 90% identity to SEQ ID NO: 1; and / or a light chain variable domain having a sequence with at least 90% identity to SEQ ID NO: 2.

[0275] The sequence identity of at least 90% identity of the above preferred embodiment and als in general herein at each occurrence independently is with increasing preference at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity and 100% identity.

[0276] In a preferred embodiment of the third aspect the antigen binding protein comprises or is an antibody, a SCGF-binding fragment of an antibody or an antibody-like protein.

[0277] In a more preferred embodiment of the third aspect

[0278] (i) the antibody is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, or a rodent, in particular a mouse antibody or from any other species; and / or

[0279] (ii) the antibody is a mono-specific, bi-specific, tri-specific, or multi-specific antibody; and / or

[0280] (ii) the antibody is a single chain antibody, a single chain variable fragment antibody, a diabody, a Fab fragment, or an F(ab)2 fragment, single-chain antibody (VH-only antibodies) or a nanobody; and / or

[0281] (iv) the antibody is selected from an IgA, IgE, IgG, in particular IgGl, an IgG2 antibody, an IgG3 antibody, and an IgG4 antibody; and IgM; and / or

[0282] (v) the antibody-like protein is selected from the group consisting of lipoprotein-associated coagulation inhibitor (LACI-D1); affilin, e.g. human-γ B crystalline or human ubiquitin; cystatin; Sac7D from Sulfolobus acidocaldarius; lipocalin, anticalin; designed ankyrin repeat domains (DARPins); SH3 domain of Fyn; Kunits domain of a protease inhibitor; monobodies, e.g. the 10th type III domain of fibronectin; adnectins: cysteine knot miniproteins knottins; atrimers; evibodies, e.g., affibodies, e.g. three- helix bundle from Z-domain of protein A from Staphylococcus aureus; Trans-bodies, e.g. human transferrin; tetranectins, e.g. monomeric or trimeric human C-type lectin domain; microbodies, e.g. trypsin-inhibitor-II; an armadillo repeat protein; tetraspanins, B-type lection domain containing elements, EZH type domain containing elements, avimers, nanofitins and affilins.

[0283] In a preferred embodiment of the third aspect the antigen binding protein has at least one of the following characteristics:

[0284] a. binds to SCGF with a Kdof less than 1 nM, of less than 100 pM, of less than 10 pM, or of less than 5 pM; and / or

[0285] b. blocks the binding of SCGF to a cell responsive to SCGF with an IC50of less than 1 nM, preferably less than 200 pM; and / or

[0286] c. triggering cytokine changes in tumor-associated macrophages with an EC50of 0.1 – 1 μg / ml in vitro. In a preferred embodiment of the first, second and third aspect the antigen binding protein further comprises at least one compound selected from:

[0287] (i) a second antigen binding protein specifically binding to a target different from SCGF, preferably to the ectodomain of a different protein than SCGF, wherein the target different from SCGF is preferably an immune checkpoint protein (such as PD1, PDL-1 or CTLA-4), CCR5, HGF receptor, or an IGF-1 receptor;

[0288] (ii) a second antigen binding protein specifically binding to an SCGF receptor;

[0289] (iii) a toxin,

[0290] (iv) a label, preferably a radionuclide or a fluorophore;

[0291] (v) a polypeptide comprising or consisting of a transmembrane domain and, optionally an endo domain,

[0292] (vi) a drug,

[0293] (vii) a chemokine,

[0294] (viii) a cytokine,

[0295] (ix) an enzyme,

[0296] (x) a component modulating serum half-life, and / or

[0297] (xi) an Fc part of an antibody.

[0298] In a preferred embodiment of the first, second and third aspect the antigen binding protein comprises an scFv fragment being fused to an Fc part, wherein the Fc part is preferably a human IgGl or mouse IgG2 Fc part and / or a genetically engineered Fc part that abrogates binding of Fc receptors.

[0299] In a fourth aspect the present invention provides a nucleic acid or a set of two nucleic acid molecules encoding the antigen binding protein of the first, second or third aspect of the invention is provided.

[0300] In a fifth aspect the present invention provides a recombinant expression vector or a set of two recombinant expression vectors comprising a nucleic acid molecule(s) according to the fourth aspect of the present invention.

[0301] In a sixth aspect the present invention provides a host cell comprising the vector or set of two vectors of the fifth aspect of the invention.

[0302] In a seventh aspect the present invention provides a method of making the antigen binding protein of the first, second or third aspect of the invention, comprising the step of preparing said antigen binding protein from a host cell expressing said antigen binding protein. In an eighth aspect the present invention provides a pharmaceutical composition comprising at least one antigen binding protein according to the first, second or third aspect of the invention, the nucleic acid according to the fourth aspect of the invention, or the vector according to the fifth aspect of the invention, or the host cell of the sixth aspect of the invention, or a combination thereof and optionally further comprising a pharmaceutically acceptable excipient.

[0303] In accordance with a preferred embodiment of the eight aspect the pharmaceutical composition further comprises one or more compounds being selected from

[0304] (i) an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is preferably an antibody or a small molecule against PD1, PDL-1 or CTLA-4 and is most preferably Nivolumab,

[0305] (ii) an inhibitor of the chemokine receptor CCR5, wherein the CCR5 inhibitor is preferably Maraviroc,

[0306] (iii) anti-tumor lymphocytes, wherein the anti-tumor lymphocytes are preferably chimeric antigen receptor T-cells (CAR T-cells), T-cell-receptor-engineered T-cells (TCR T- cells), chimeric antigen receptor NK-cells (CAR NK-cells), NK cell receptor- engineered NK cells (NCR NK-cells), TCR / CAR hybrid T-cells, NCR / CAR hybrid NK-cells or tumor-infiltrating lymphocytes (TILs), and

[0307] (iv) a tyrosine kinase inhibitor, wherein the tyrosine kinase inhibitor is preferably an inhibitor of the HGF receptor and is most preferably Capmatinib.

[0308] Regarding item (i) it is noted that Figure 25 shows that anti-SCGF treatment synergizes with immune checkpoint blockade (Nivolumab treatment). Regarding item (ii) it is noted that Figure 26 shows that anti-SCGF and maraviroc treatment synergize in solid tumours. Regarding item (iii) it is noted that Figure 23 shows a synergy of anti-SCGF treatment and Capmatinib-mediated HGF receptor inhibition.

[0309] In a ninth aspect the present invention provides a kit for the treatment of cancer, inflammation, or trauma comprising at least one antigen binding protein according to first, second or third aspect of the invention, the nucleic acid according to the fourth aspect of the invention, the vector according to the fifth aspect of the invention, the host cell of the sixth aspect of the invention, the pharmaceutical composition of the eighth aspect of the invention, or a combination thereof.

[0310] In a tenth aspect the present invention provides a SCGF antagonist for use in treating or preventing a condition associated with cancer, inflammation and / or trauma in a subject.

[0311] In a preferred embodiment of the tenth aspect of the invention the SCGF antagonist is selected from the group consisting of: (i) antibodies and SCGF-binding fragments thereof,

[0312] (ii) antibody-like proteins,

[0313] (iii) inhibitory variants of SCGF;

[0314] (iv) inhibitors of SCGF-activity;

[0315] (v) a nucleic acid encoding (i) to (iii); and

[0316] (vi) a vector comprising (v).

[0317] In a preferred embodiment of the tenth aspect of the invention the inhibitors of SCGF-activity is selected from the group consisting of:

[0318] (i) an oligonucleotide that specifically binds to SCGF, preferably a DNA-aptamer, D-RNA aptamer, or a L-RNA aptamer; or

[0319] (ii) an oligonucleotide selected from the group consisting of antisense DNA, antisense RNA, siRNA, and miRNA.

[0320] In a preferred embodiment of the tenth aspect of the invention the SCGF antagonist is at least one antigen binding protein according to first, second or third aspect of the invention, the nucleic acid according to the fourth aspect of the invention, the vector according to the fifth aspect of the invention, the host cell of the sixth aspect of the invention, the pharmaceutical composition of the eighth aspect of the invention, or a combination thereof.

[0321] In a preferred embodiment of the tenth aspect the cancer comprises cancer stem cells (CSC). In a preferred embodiment of the tenth aspect the cancer is resistant to standard therapy, preferably, wherein the cancer is resistant to chemotherapy and / or immunotherapy and / or radiation therapy.

[0322] In a preferred embodiment of the tenth aspect the cancer is a solid tumour.

[0323] In a preferred embodiment of the tenth aspect the cancer is selected from ovarian cancer, colorectal cancer, pancreatic cancer, lung cancer, renal cancer, bladder cancer, prostate cancer, breast cancer, leukemia, myeloma, and skin cancer. More preferred the cancer is selected from ovarian cancer or colorectal cancer.

[0324] In a preferred embodiment of the tenth aspect the inflammation is selected from pulmonary fibrosis, multiple sclerosis, auto-immune diseases (lupus erythematosus, lichen planus, rheumatoid arthritis, Sjogren syndrome, Morbus Bechterew, primary sclerosing cholangitis, ulcerative colitis, Morbus Crohn, mesangioproliferative nephritis, Guillain-Barre-Syndrome, rheumatic fever, Hashimoto thyreoiditis and Morbus Still, Morbus Basedow, Diabetes mellitus type 1, Myasthenia gravis, idiopathic thrombocytopenia, celiac disease and sarcoidosis. In a preferred embodiment of the tenth aspect the trauma can be of any mechanical, physical or chemical trauma to tissues or organs (e.g. traffic accident, stroke, cardiac infarction, pulmonary embolism).

[0325] In a eleventh aspect the present invention provides at least one antigen binding protein according to first, second or third aspect of the invention, the nucleic acid according to the fourth aspect of the invention, the vector according to the fifth aspect of the invention, the host cell of the sixth aspect of the invention, the pharmaceutical composition of the eighth aspect of the invention, or a combination thereof and optionally in addition one of the further compounds being selected from items (i) to (iv) of the preferred embodiment of the eight aspect for use in treating or preventing a disease, wherein the disease is preferably a cancer, an inflammatory disease and / or a trauma in a subject, wherein the subject is preferably an adult.

[0326] In a preferred embodiment of the tenth and eleventh aspect the cancer is resistant to standard therapy, preferably, wherein the cancer is resistant to chemotherapy and / or immunotherapy and / or radiation therapy.

[0327] In a preferred embodiment of the eleventh aspect the cancer is a solid tumour.

[0328] In a preferred embodiment of the eleventh aspect

[0329] (i) the cancer is selected from ovarian cancer, colorectal cancer, pancreatic cancer, lung cancer, renal cancer, bladder cancer, prostate cancer, breast cancer, leukemia, myeloma and skin cancer, preferably ovarian cancer or colorectal cancer;

[0330] (ii) the inflammatory disease is selected from pulmonary fibrosis, multiple sclerosis, autoimmune diseases (lupus erythematosus, lichen planus, rheumatoid arthritis, Sjogren syndrome, Morbus Bechterew, primary sclerosing cholangitis, ulcerative colitis, Morbus Crohn, mesangioproliferative nephritis, Guillain-Barre-Syndrome, rheumatic fever, Hashimoto thyreoiditis and Morbus Still, Morbus Basedow, Diabetes mellitus type 1, Myasthenia gravis, idiopathic thrombocytopenia, celiac disease and sarcoidosis; and / or

[0331] (iii) the trauma is a mechanical, physical or chemical trauma to tissues or organs (e.g. traffic accident, stroke, cardiac infarction, pulmonary embolism).

[0332] In a preferred embodiment of the tenth and eleventh aspect the cancer is a hematologic malignancy.

[0333] The examples illustrate the claimed invention, Examples

[0334] Concepts and ideas

[0335] SCGF has been connected to inflammation, CSC proliferation, relapse and metastasis in cancer, making it an important target for therapy. While we know some of the functions of SCGF in cancer, its cellular origin within solid tumours and ovarian cancer in particular is still largely unknown. Northern blot analysis revealed that the mRNA for SCGF is detected in myeloid cells and fibroblasts, but not in lymphocytes (Hiraoka et al, 1997). It is expressed in the monocytic lineage THP-1 cell line, in the spleen and in immature neutrophils, but not in myeloblasts, mature neutrophils or extracellular bone marrow fluid (Perrin et al, 2001).

[0336] Given the role of SCGF in promoting proliferation of CSCs, we were interested in whether SCGF changed stem cell markers in ovarian tumours and the tumour microenvironment. This microenvironment also contains immune cells, cancer cells, fibroblasts and endothelial cells that all play a role in cancer progression.

[0337] Because SCGF is involved in pro-inflammatory responses and promotes the proliferation of CSCs, we suspect that TAMs might mediate these functions in part by secreting SCGF. In our laboratory, macrophages isolated from pleural effusions in breast cancer were found positive for SCGF as evaluated by immunohistochemistry. This preliminary evidence reinforces our hypothesis that macrophages might secrete SCGF in the tumour microenvironment of ovarian cancer.

[0338] Cancer Associated Fibroblasts (CAFs) are emerging as a target for antibodies, vaccines and chimeric T-cells. However, given the role of CAFs in inflammation, chemo-resistance and maintenance of the CSC niche, it is conceivable that some of these functions are mediated by SCGF. SCGF mRNA has indeed been identified in fibroblasts (Hiraoka et al, 1997). Furthermore, in gastrointestinal stromal tumours, the stroma is SCGF-positive (Da Riva et al, 2011). Therefore, without wishing to be bound by theory we hypothesize that FAPa positive CAFs were also expressing SCGF in the stromal microenvironment of ovarian cancer.

[0339] The inventors cloned and purified antibodies against human SCGF. Three different antibodies were developed, two scFv fragments linked to human IgGl or mouse IgG2a and one full length chimeric antibody. The scFv antibodies produced here are modified from a normal scFv fragment. scFv antibodies are not normally linked to Fc portions. We included this aspect to the design to increase the size of the antibody and include the FcRn binding site. FcRn binding increases half-life from a few hours to a few weeks (Chames et al, 2009). We also included a mutation at Asn297 (N297Q) to prevent interaction with FcyR and Clq so the function is not influenced by other immune cells. This aglycosylation ensures that the effect produced by our antibody is a direct consequence of antigen binding. Aglycosylated antibodies are currently being tested in different clinical trials and it has been observed that the mutation does not reduce antigen binding affinity, stability at physiological or low temperatures, pharmacokinetics and bio distribution (Ju and Jung, 2014). Niwa et al. found that the mutation to Asn297 resulted in increased cytotoxic activity of mouse and all human IgG antibody isotypes. The full length antibody is not modified at Asn297 and will be able to engage in FcR binding however it is much larger and may not be able to penetrate the tumour as efficiently as the scFv antibodies. However the FcR mediated functions may be essential for overall anti-tumour effect therefore by comparing both antibodies we will be able to assess how to inhibit SCGF most efficiently.

[0340] Materials and Methods

[0341] Explant Tissue Culture and Patient Characteristics

[0342] Ovarian carcinoma tissue was obtained from the Universitatsfrauenklinik Heidelberg with written consent from all patients prior to analysis. The experiments conducted on the tissue samples were approved by the medical ethics committee of Heidelberg University. All tissues were collected during exploratory surgery before treatment. The patients were between 42 and 78 years in age. Additional clinical details of the patients are recorded in Table 1.

[0343] Table 1: EOC patient characteristics (w = 40).

[0344] Characteristic Value

[0345] ■■

[0346] Mean 60

[0347] Range 42-78

[0348] l it mon r lx pe

[0349] High grade serous (HGSOC) 27

[0350] Low grade serous (LGSOC) 3

[0351] Endometrioid 2

[0352] Carcinoma, little differentiated 2

[0353] Clear cell carcinoma 1

[0354] Mucinous 1

[0355] Unknown 4

[0356]

[0357] The resected tissue was quickly transferred in sodium chloride to the laboratory where it was processed under a sterile cell culture hood as described previously. Directly upon reception, a portion of each tissue was either stored frozen in OCT or formalin-fixed, paraffin embedded (FFPE). For tissue culture, two sections were placed into each well of a 24 well plate with 1ml medium. The tissue was cultured in medium containing 1XMEM (10X MEM Gibco 21430020) with 1% L-glutamine (Sigma Aldrich G7513) and 7.5% Sodium bicarbonate (Roth HN01.1) at a pH of approximately 7.4. The plates were cultured on a shaker in a Whitley H35 hypoxystation with 15% 02, 5% CO2 and 85% N2. Some sections were treated with hybridoma supernatant containing anti-SCGF antibody (Aldevron) or the ScFv-hlgGl anti-SCGF antibody that we produced. Culture was stopped at 24, 48 and 72 hours after the initial processing. The number of time points depended on the size of the tissue received. At each time point, the tissue sections were collected for cryopreservation and paraffin embedding.

[0358] T cell migration experiments

[0359] T cells (either autologous TILs expanded ex vivo or CAR T cells) were stained with 5 pM CMFDA for 1 h and cryopreserved in FBS (Biochrom) with 10% DMSO prior migration experiment. The tissue explants were treated with anti-SCGF antibody (clone 8326.1) at a concentration of 5 pg / ml 1 hour before CMFDA T cell addition. At CMFDA-T cell addition, the medium was replaced by fresh medium, the tissue was treated one more time with the antibody at 1 million CMFDA T cells were added on each explant. After 24h, tissues were harvested and either stored at -80°C in OCT or formalin fixed, paraffin embedded.

[0360] Cell culture

[0361] For TAM isolation, malignant ascites from EOC or breast cancer patients were collected and centrifuged at resuspended at a density of 5 million cells per ml in a T75 flask for differential adherence. The cells were left to adhere for 90 minutes, then the non-adherent cells were washed away and the adherent TAMs were cultured in a medium composed of ascites supernatant and RPMI 1640 (Gibco) with 1% glutamine at a 1:1 ratio until further use.

[0362] Monocytes were isolated from the blood of healthy volunteers. PBMCs were isolated using Lymphoporep (Stemcell) gradient centrifugation for 30 minutes at 400g with no brake, followed by two wash steps in RPMI and one slow centrifugation (100g for 10 minutes with no brake) to deplete platelets. CD 14+ monocytes were isolated by magnetic cell sorting with CD 14 microbeads (Miltenyi Biotec) following the manufacturer’s instructions and the purity was assessed by a CD 14 immunostain on a cytospin. For monocyte- to-macrophage differentiation, the cells were cultured in RPMI 1640 (Gibco) + 1% glutamine + 10% foetal calf serum (FCS) and treated 50 ng / ml M-CSF (Peprotech #300-25) every two days for one week. For differentiation into TAMs, the monocytes were cultured in RPMI1640 and malignant ascites supernatant (1:1 v:v) for 48 hours. The differentiation was confirmed by immunostaining for CD 163 and by observation of the typical morphological changes (TAMs are oblong and bigger). Monocytes, macrophages and TAMs were treated with anti-SCGF at 5 pg / ml for 18 hours prior harvesting of the supernatant and protein lysis for subsequent protein analysis.

[0363] Stains, Immunohistochemistry (IHC) and Immunofluorescence (IF)

[0364] Tissue sections were prepared from formalin fixed, paraffin embedded tissue (4pm) or cryosections (6pm). The cryosections were fixed in either 4% paraformaldehyde (Roth P087.3) or 33% acetone in methanol prior to staining. The sections were analysed for the presence and spatial distribution of immune cell markers (CD3, CD8, Granzyme B, CD163) and other specific markers (CAI 25, CD 105, FAPa, EpCAM, activated caspase 3). Staining conditions and antibody references are listed in Table 2. The staining procedures were carried out on a Leica BOND Max automatic staining machine. This is a biotin-free detection system that contains a peroxidase block, post primary antibody, polymer reagent, DAB chromogen and haematoxylin counterstain (BOND polymer refine detection kit Leica DS 9800). The machine also performs antigen retrieval procedures as specified by the user. First, endogenous peroxidases are blocked. After blocking, the primary antibody is added. Then the post primary rabbit anti-mouse IgG polymer localizes primary mouse antibodies. The polymer reagent is an anti-rabbit HRP IgG that recognizes the post primary antibody and amplifies the signal. DAB is then used to visualize the staining along with a haematoxylin counterstain. Once the automatic staining procedure was completed, the slides were mounted with Aquatex (Merck Millipore 108562) and scanned using the Leica Aperio AT2 whole slide scanning machine or the NanoZoomer S60 (Hamamatsu) slide scanner at a magnification of 20X, or of 40x in case of a double staining (EpCAM-activated caspase 3).

[0365] Immunofluorescent stains were carried on cytospin sections of tumour-associated macrophages from breast cancer or ovarian cancer ascites. Cells were collected by scraping and spun at 5000g for 5 minutes onto SuperfrostPlus slides (R Langenbrinck 03-0060) in a cytocentrifuge, at a density of 200.000 cells per 200 pl per each slide. The sections were fixed with 4% paraformaldehyde and the primary antibody was incubated overnight. The secondary antibody was incubated for an hour. The slides were mounted using DAPI Fluoromount-G Medium (Southern Biotech 0100-20) and scanned on the Nanozoomer S60 scanner (Hamamatsu) at a magnification of 20X.

[0366] Masson trichrome stains ware carried on 6 pm-thick FFPE sections using the Masson trichrome kit (Sigma- Aldrich, HT15) and Weigerts Hematoxylin for counterstain.

[0367] Table 2: Description of Antibodies used for IHC-P and IHF / IF.

[0368] Antibody Clone Reference

[0369] II IC-P

[0370] CD3 Sp7 Abeam #ab 16669

[0371] CD8 4B11 Novocastra # NCL-CD8-4B1 1 CD 163 EDHu-1 AbD Serotec #MCA1853

[0372] Granzyme B 23H8L20 Invitrogen #701395

[0373] Cleaved caspase 3 Polyclonal Abeam #Ab2302

[0374] EpCAM D1B3 Cell Signalling #2626

[0375] CD 105 Polyclonal Thermo Scientific ##PA5-16895 11 IC-fro / .en tissue c r c\ lospm or

[0376] CD 163 EPR1951 Abeam #ab 182422

[0377] 8

[0378] CLEC5A polyclonal R& D #AF2384

[0379] FAPalpha Polyclonal Biorbyt #orb227989

[0380]

[0381] Immune Cell Quantification and Image Analysis

[0382] The number of stained immune cells (CD3, CD8, CD163, Granzyme B) and stained blood vessels (CD105) was quantified from the whole slide images. These were complete microscopic images of full tissue sections. Regions of the tissue were manually drawn to remove any artefacts and necrotic or fatty regions within the tissue. Cell counts were generated using specially developed software programs in Visiopharm (VIS software suite) or Halo (Indicalabs) and the number of cells per square millimetre was quantified. Separate algorithms were designed for each marker. These algorithms detected and counted the stained cells using different parameters such as size, shape and colour. This technology has been previously reported (Halama et al, 2009, Halama et al, 2011). For double stains for EpC AM-activated caspase 3, the Halo software (Indicalab) was used and a specific algorithm was designed and validated to detect the total EpCAM-positive cancer cells and the EpCAM-positive activated caspase 3 -positive, dying tumours cells.

[0383] Stainings from IHC (CD68, SCGF, FAPa) were used to create virtual overlay images. The automated workflow used is based on deformable image registration exploiting the Markov random field formulation and discrete optimization algorithms (Glocker et al, 2011), removal of histological noise through quaternionic operations (Wu et al, 1999) and 2D histogram variance thresholding (Valous et al, 2013), colour deconvolution for stain separation (Ruifork and Johnston, 2001), colour / brightness / saturation geometric transformations in a Clifford algebra framework (Batard et al, 2009), and post-processing operations. In this in silico approach, we used standard IHC methods to visualize the distribution of multiple biomarkers in a single virtual section.

[0384]

[0385] ;x Protein

[0386] Protein lysates were prepared from frozen tissue using the Bioplex tissue lysis kit (Bio-Rad Laboratories) according to the manufacturer’s instructions. The amount of protein was quantified using a BCA protein assay kit (Thermo Scientific).

[0387] The protein samples were analysed for 50 cytokines and chemokines: CTACK-CCL27, GROa, HGF, ICAM-1, IFN-a 2, IL-2 R-a, IL-3, IL-12p40, IL-16, IL-18, LIF, MCP-3 CCL7, M-CSF, MIG CXCL9, 0-NGF, SCF, SCGF-0, SDF-la CXCL12, TNF-0, TRAIL, VCAM-1, IL-la, MIF, IL-ip, IL-lra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, Eotaxin, FGF basic, G-CSF, GM-CSF, IFN-g, IP-10, MCP-1, MIP-la, MIP-ip, PDGF bb, RANTES, TNF-a and VEGF using pre-designed panels (Bio-Plex Pro Human Cytokine 21-Plex Panel, Bio-Plex Pro Human Cytokine 27-Plex Panel, Bio-Plex Pro Human ICAM-1 Assay, Bio-Plex Pro Human VCAM-1 Assay, Bio-Rad Laboratories). The Luminex xMAP technology from Bio-Rad uses color-coded capture beads conjugated with monoclonal antibodies. Each bead is labelled with two fluorophores (excited by a red laser) that create different bead regions according to the measured intensities. Biotinylated detection antibodies recognize the different analytes which are visualized with a Streptavidin-Phycoerythrin (SA-PE) reporter by excitation of the green laser. In an additional set of experiments, the protein samples or cell culture supernatants were analysed for other cytokines: APRIL / TNFSF13, BAFF / TNFSF13B, sCD30 / TNFRSF8, sCD163, Chitinase-3 -like 1, gpl30 / sIL-6RP, IFN-a2, IFN-P, IFN-y, IL-2, sIL-6Ra, IL-8, IL-10, IL-11, IL-12 (p40), IL- 12 (p70), IL- 19, IL-20, IL-22, IL-26, IL-27 (p28), IL-28A / IFN-X2, IL-29 / IFN-M, IL-32, IL-34, IL-35, LIGHT / TNFSF14, MMP-1, MMP-2, MMP-3, Osteocalcin, Osteopontin, Pentraxin-3, sTNF-Rl, sTNF-R2, TSLP, TWEAK / TNFSF12, using a predesigned panel (Bio-Plex Pro™ Human Inflammation Panel 1, 37-Plex) and following the same procedure.

[0388] The manufacturer’s protocol was followed while preparing the plate. The standard curve was prepared as a fourfold standard dilution series. The color-coded magnetic capture beads were diluted and added to the 96-well assay plate. The plate was washed twice before samples (300pg / ml protein), standards and blank were added. The plate was incubated in the dark on a shaker at 900rpm for 30 minutes. After three washes, the biotinylated detection antibodies were diluted and added. The plate was incubated again under the same conditions before washing. SA-PE reporter reagent was incubated for 10 minutes before washing. The beads were resuspended and the plate was shaken for 30 seconds. The analysis was carried out on a Bio-plex reader with a 532 nm reporter laser and 635 nm classification laser (Bio-Rad Laboratories). Cytokine concentrations (pg / ml) were calculated with Bio-Plex Manager 4.0 software by optimizing the standard curves for each cytokine.

[0389] Antibody preparation

[0390] The laboratory had 3 hybridomas (clone 1E4, 8B6 and 9F6) from Aldevron that produced anti-SCGF antibodies in the supernatant. The hybridoma supernatants were all tested in preliminary experiments (IHC, ELISA and Luminex cytokine analysis). However, when these hybridomas were sequenced, they all contained the same sequence. Dr. Frank Momburg designed a cDNA sequence that contained the heavy and light chains along with restriction sites for further cloning. This synthetic cDNA sequence was produced by Genescript. From this sequence, three antibodies were prepared. Two scFv fragments ligated to mouse-IgG2a and human-IgGl (H-CH2-CH3) along with a full length chimeric antibody containing human IgGl (CH1-CH2-CH3) and human Ig kappa. The scFv antibodies were aglycan with a mutation (N297Q) in the CH2 domain of IgGl that prevented glycosylation and interaction with other Fc portions. The full antibody was prepared by cutting out the heavy and light chains from the sequence. The light chain was ligated to human Ig kappa and the heavy chain was ligated to human IgGl (CH1-CH2-CH3). A description of the sequence is shown in figure 3 below.

[0391] a) Cloning

[0392] All restriction digests were carried out in a total volume of 50pl for 90 minutes followed by 1 pl fastAP (Thermo Scientific EF0651) for dephosphorylation (30 minutes). After restriction, the fragments were run on a 1% agarose gel at 130mA for 30 minutes. The correct fragment was cut out under a UV light. The DNA was extracted using the QIAquick gel extraction kit (QIAGEN 28706) according to the manufacturer’s protocol. The vector and the insert were ligated using T4 DNA ligase (Thermo Scientific EL0012) and 10X ligaseB (Thermo Scientific) in a total volume of 20pl. The mixture was incubated overnight at 16°C. The ligation mixture was added to 50pl E.coli XLlBlue and incubated on ice for 25 minutes. This was subjected to a heat shock at 42°C for 90 seconds and then spread on agar+1% Ampicillin plates. The plates were incubated overnight at 37°C. After 24 hours, single colonies were picked and grown in 4ml LB medium + 1% Ampicillin overnight. A mini prep was performed on these colonies using the QIAprep Spin Miniprep kit (QIAGEN 27104). All constructs were confirmed by sequencing (Eurofins Genomics) before cloning into a pcDNA mammalian expression vector. Maxi preps were prepared using the Contactprep plasmid maxi kit (QIAGEN 12863) and all constructs (mouseIGg2a ScFv, humanlgGl ScFv and the full chimeric human antibody) were sequenced again. A detailed description of the cloning process along with the sequences of the plasmid and vectors is presented below.

[0393] The scFv fragment was cut out using Mfel and Xhol from the ordered cDNA plasmid (Figure 3). This was then inserted into vector 8079.1 or 8169.1. The heavy chain was obtained using EcoRl. This was inserted into 8143.1. The light chain was obtained by cutting first with Xhol and BsiWl and then EcoRl. This was inserted into 8133.1. Once the constructs were confirmed by sequencing, all constructs were cut with Xhol and Notl. The fragments were cloned into a pcDNA mammalian expression vector.

[0394] In the sequences listed hereunder, the StrepTac purification sequence is indicated in bold, the restriction sites are written in underlined italics and the stop codons are indicated by asterisks.

[0395] 8133.1 pBluescript KSII+ / hIgG K

[0396] RTVAAPSVFI FPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* * (SEQ ID NO: 12 )

[0397] 8134.1 pBluescript KSII+ / hIgGl CH1-CH2-CH3 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS WTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* * (SEQ ID NO: 13)

[0398] 8079.1 pBluescnpt KSII scFvAG5ZAmIgG2a-Fc[C224S, N297Q1-StrepTag* G 5GGGGSGGGGSGGGGSASEPRGPTIKPSPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPMVTC VWDVSEDDPDVQISWFVNNVEVLTAQTQTHREDYQSTLRWSALPIQHQDWMSGKEFKCKVNNKAL PAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTE PVLDSDGSYFMYSKLRVEKKNWVERNSYSCSWHEGLHNHHTTKSFSRTPGKDRGWSHPQFEKSR* (SEQ ID NO: 14 )

[0399] 8169.1 pBluescnpt KSII scFvAG5ZAhIgGl-Fc[C220S, N297Q1-StrepTag* G 5GGGGSGGGGSGGGGSASEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV WDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRWSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKDRGWSHPQFEKSR* (SEQ ID NO: 15)

[0400] b) Cell Culture and Transfection

[0401] i. Stable Transfection

[0402] All constructs obtained from the maxi preps were transfected into HEK 293 cells. The cells were cultured in RPMI (Sigma Aldrich RPMI 1640) + 1% L-glutamine + 10% FCS. The cells were seeded at 2X105cells per well in a 6- well plate. The transfection mix contained 4 pg DNA, 8pl peqFECT (peqlab 13-8010) and 400pl serum-reduced Optimem (Gibco 31985). The transfection mixture was incubated for 20 minutes at RT and added dropwise to the cells. After transfection, the cells were cultured in RPMI + 10%FCS + 3X Anti- Anti (Gibco 15240). After 3 days, the selection was started using Img / ml G418

[0403] ii. Transient Transfection

[0404] The constructs were transfected into CHO-S cells. The cells were cultured in PowerCHO medium (Lonza BE12-771Q). The final constructs (0.625pg DNA / million cells) were combined with PEI (2.5pg / million cells, Polysciences 23966-2). This mixture was added to the CHO-S cells at a density of 4 million cells / ml. After transfection, the cells were cultured in ProCHO medium (Lonza 12-029Q). The cells were incubated at 31°, 8% CO2 in glass bottles on a shaker at 125rpm. Two batches of CHO-S transfections were prepared. The supernatant from the CHO-S transfections were collected after 6 days in culture. c) Purification

[0405] i. Protein A purification

[0406] Beads of protein-A (ProSep-vA High Capacity, Millipore) were added to a PDlO-column (Sephadex) and the supernatant was added to the column. The protocol is shown in table 3. An OD280nm measurement (NanoDrop 2000) was obtained for each of the elution fractions. Samples were collected for a SDS-Page analysis. These included the crude supernatant (CR), the flow through (FT) and the wash pool (WP).

[0407] Table 3: Protein A purification protocol

[0408] Supernatant; 4°C. Iml / min

[0409] loading

[0410] Wash 1 4°C. 1.5 ml / min. PBS

[0411] Wash 2 4°C. 1.5 ml / min. PBS + 0.1 % Twccn20 + 0.1 mM NaCl

[0412] Wash 3 4°C. 1.5 ml / min. PBS

[0413] Elution RT. glycin-HCl. pH 3

[0414]

[0415] Fractions; 500 pl fractions in 300 pl Trisd-HCl neutralization buffer

[0416] ii. StrepTactin purification

[0417] Beads of StrepTactin were added to a PDlO-column. The supernatant was filtered using a 0.22pm PES filter before adding to the column. Biolock (3 l / ml) was added to the supernatant to block any active biotin. This protocol is shown in Table 4. Samples were collected for a SDS-Page analysis. These included the crude supernatant (CR), the flow through (FT) and the wash pool (WP). Table 4: StrepTactin purification protocol

[0418] Supernatant RT. 1 ml / min

[0419] loading

[0420] Wash 1 RT. 1.5 ml / min. 30 ml wash buffer lx

[0421] Elution RT. 5 ml elution buffer

[0422]

[0423] Fractions 500 pl fractions

[0424] The CR, FT, WP and elution fractions with the highest absorbance values were run on an SDS-Page gel. The samples (5 pg protein from the elution fractions) were combined with loading buffer (4X Runblue LDS) and IX DTT. Each elution fraction was also run as a non-reducing condition without DTT. The samples were loaded onto the gel (Expedion 10% SDS) and run for 90 mins at 140V. The gel was stained with Instant Blue (Expedion) and washed before pictures were taken.

[0425] After verification through SDS-Page, the elution fractions with the highest absorbance values from the protein A purification were loaded into a spin concentrator (Amicon Ultra, Millipore) with a lOKDa molecular weight cut off and centrifuged at 13,200 rpm until the samples were concentrated at a final volume of 150pl. The elution fractions from the StrepTactin purification were loaded into a Vivaspin-6 concentrator with a lOKDa molecular weight cut off and centrifuged at 4000rpm until the samples were concentrated at a final volume of 300pl.

[0426] d) Confirmation of binding efficiency using ELISA

[0427] A capture ELISA was used to measure the binding efficiency of our new antibodies compared to the hybridoma supernatant (clone 9F6) from Aldevron. We chose this clone because it has produced the strongest results in previous IHC and tissue culture experiments. A 96 well plate was coated with 60ng Glutathione Casein (GC) and incubated at 4°C overnight. The wells were blocked with blocking buffer for 1 hour. The wells were then coated with recombinant SCGF at 1 pg / well for 1 hour. This recombinant antibody had a tag for GC binding. Each of our new anti-SCGF antibodies were tested at 3 different concentrations. We also tested the hybridoma supernatant, our new nonpurified supernatant and a GC tagged positive control. After incubation for an hour with the primary antibodies, the secondary antibodies were added. Two different secondary antibodies were used, a goat anti-mouse HRP and a goat anti-human HRP (1:10000). All incubations were carried out on a shaker at room temperature. For the detection we used OPD with 3% H2O2. The plate was incubated in the dark for 30 minutes and the reaction was stopped with H2SO4. The measurement was done at 490nm on an ELISA reader (TECAN). The following ELISA buffers were used: Coating Buffer (pH 9.6): 10ml 0.05MNa2C03, 40 ml 0.05MNaHC03(for 50ml buffer); Blocking Buffer: PBS, 0.05% Tween20, 0.2% Casein; Wash Buffer: PBS, 0.1% Tween, 0.2% Casein.

[0428] Results

[0429] All three anti-SCGF antibodies display the right size on SDS Page gels after purification:

[0430] Three anti-SCGF antibodies were cloned and purified; scFv-msIgG2a (i.e. scFv fragment that was ligated to mouse IgG2a), scFv-hlgGl (i.e. scFv fragment that was ligated to human IgGl (CH2-CH3)) and one full length anti-SCGF antibody. Once all the antibodies were purified, SDS Page gels were used to confirm the size of the antibody under reducing conditions. DTT was used to reduce the disulphide bridges so that the antibodies could adopt a random coil conformation and separate in the SDS Page gel. We also ran the antibodies under non-reducing conditions and a clear difference could be observed between the two conditions. All the elution fractions from the purification were quantified using a spectrophotometer (260nm) and the fraction with the highest amount of protein was run in the SDS Page gels. In figure 4A, the mouse and human scFv anti-SCGF antibodies were run along with the crude supernatant (CR), flow through (FT) from the column and wash pool (WP). These antibodies were produced in CHO-S cells and purified using protein A. In all the lanes, except the non-reducing condition, a band at around 50KDa was observed. This corresponds to the size of the expected antibodies and only this band was observed after the purification. In the gel from figure 4B, the full length antibody was run. This antibody was produced in CHO-S cells (transient transfection) and HEK cells (stable transfection) as a double transfection with both the heavy and light chain plasmids. The antibodies were purified using protein A. The CR, FT, WP and elution fraction with the highest protein measurement were run on the SDS Page gel. In all the lanes, except the non-reducing conditions, two bands were observed at around 50KDa and 28KDa. These bands remained after purification and were present on both gels from both transfections. This confirms the presence of both heavy (52KDa) and light chains (23.5KDa).

[0431] All three anti-SCGF antibodies are able to bind to recombinant SCGF efficiently in a capture ELISA Once we were able to purify and confirm the size of the antibodies, we tested their binding capacity using ELISA. The ELISA plate was coated with SCGF containing a GC tag that bound to the GC coated plates. The binding affinity of the new antibodies was compared to the affinity of the initial mouse hybridoma supernatant and the non- purified supernatant from the transfections. The hybridoma supernatant was tested at a dilution of 1:200. This dilution was established in previous ELISA experiments. The reaction was observed using HRP conjugated secondary antibodies and absorbance was measured at 490nm. All the purified antibodies showed high binding efficiencies that were much higher than the supernatants (Figure 5A). Three different concentrations were tested for each antibody (1μg / ml, 2.5μg / ml and 5μg / ml) and the binding was high and did not change much. This indicates that the reaction was saturated and lower dilutions need to be used to establish a binding curve for each antibody.

[0432] The anti-SCGF antibody binds to the C-lectin-type domain of SCGF

[0433] Epitope mapping was conducted to accurately define the binding site for the antibody on recombinant SCGF. For this purpose, the binding of the scFv-mIgG2a anti-SCGF antibody was tested on overlapping 15mer peptides covering the whole SCGF sequence and the results clearly indicated that the anti-SCGF antibody specifically binds the C-lectin-type domain (Figure 5B). The scFv-mIgG2a anti-SCGF antibody produces strong staining in cryo-sections of ovarian tissue and the staining pattern is similar to that seen with the hybridoma supernatant:

[0434] To test the function of the mouse scFv anti-SCGF antibody we performed IHC on 5pm-thick cryosections of ovarian tissue. Consecutive sections were stained with purified scFv-mIgG2a anti-SCGF antibody (1:500) and hybridoma supernatant (clone 9F6, 1:200). Staining in 3 different tissues demonstrated that both antibodies produced a similar staining pattern (Figure 6). The scFv-mIgG2a anti-SCGF antibody produced staining with less background and a stronger signal. All staining was conducted on the BOND automatic staining machine with the same staining protocol for both antibodies. A manual peroxidase block was performed to prevent staining of endogenous peroxidase. This experiment was repeated for 5 other tissues and the staining patterns were similar.

[0435] Cytokine measurement in a tumour explant model shows a distinctive pattern of cytokine changes after treatment with anti-SCGF antibodies:

[0436] To test the function of the scFv-hIgG1 anti-SCGF antibody, two ovarian cancer tissue explants were treated with the antibody and a cytokine measurement was obtained. Two different concentrations of the antibody were tested (2.5pg / ml and 5pg / ml). These two tissues were also treated with the hybridoma supernatant. After collection at 24 and 48 hours, the tissues were cryoembedded and lysed. A cytokine measurement of the lysates using Luminex revealed a pattern of cytokine changes. In the tissues treated with both hybridoma supernatant and scFv-hIgG1 anti-SCGF, the pattern of cytokine changes was the same and the SCGF levels were decreased. In Ovar47, at 24 hours, there was a decrease in SCGF-b, VCAM-1, IL-8, IL-lra, HGF, CXCL12, IL-6, SCF, CCL7, LIF and beta-NGF (Figure 7). In Ovar0045, anti-SCGF blockade triggered a different series of effects, which were consistent between the hybridoma supernatant treatment and the scFv-hIgG1. In that tissue, a series of inflammatory factors (IL-6, IL-8, GROa, IL- 1 beta), markers of macrophage activation (MIP-la, MIP-lb) and markers of the activation of the anti-tumour immune response (IFNg, TNFa) were elevated (Figure 7).

[0437] High SCGF levels do not correlate with higher blood vessel density or immune cell infiltration:

[0438] From the cytokine measurement, we observed a decrease in proangiogenic and chemotactic factors on treatment with anti-SCGF antibodies. To test whether there was a correlation between SCGF levels and blood vessel density or immune cell infiltration, we stained direct, untreated tissue sections with CD 105, CD3, CD 8 and CD 163. These were sections from paraffin-embedded tissues cut to 4pm sections and stained with IHC. The number of cells per mm2was calculated using the Visiopharm software. There is no correlation between initial SCGF levels and immature blood vessel density stained by CD105 (Figure 8A). There is no correlation between initial SCGF levels and CD8+T-cell density in the tissues that were stained (Figure 8B). The tissues could be placed into two groups when SCGF levels were compared to CD3+T-cell density. There were those with low SCGF, low CD3+T-cells and those with low SCGF and high CD3+T-cells (Figure 8C). A similar trend was observed with SCGF compared to CD163+macrophage density. There are two groups of tissues, those with low SCGF, low CD163+macrophages and those with low SCGF and high CD163+macrophages (Figure 8D).

[0439] SCGF is

[0440]

[0441] isolated from ovarian cancer and breast cancer ascites:

[0442] Since data from the literature describes SCGF as secreted by macrophages (Gilpin et al. 2013), we stained the macrophages isolated from breast cancer effusions to evaluate SCGF expression. The macrophages from the effusions were allowed to adhere and the rest of the supernatant was washed away before cytospin slides were prepared and stained with the hybridoma supernatant (9F6, 1:200). There were many SCGF positive cells and SCGF seemed to be present at the plasma membrane and the cytoplasm. There was a difference in expression levels in different cells. There were some cells that were strongly positive and some that were weaker (Figure 9). Both Ml and M2 polarized macrophages are found in cancer. To test whether SCGF was selectively expressed by one subpopulation we established an immunofluorescence double staining with CD 163 and SCGF and CLEC5A and SCGF on the macrophages from the pleural effusions. Gonzalez-Dominguez et al. revealed CLEC5A as a marker for Ml macrophages (pro-inflammatory) and CD 163 as a marker for M2 macrophages (pro-tumour, anti-inflammatory). Double positive cells were observed when macrophages were stained with CLEC5A and SCGF. Almost all the cells were double positive but the level of SCGF was variable in different cells (Figure 10A). Double positive cells were also observed when macrophages were stained with CD 163 and SCGF. There were also some single positive SCGF cells indicating that some other cell type was also producing SCGF (Figure 10B). These preliminary data suggest that tumour-associated macrophages independent of their polarization state produce SCGF in the tumour microenvironment.

[0443] FAP+ cancer associated fibroblasts may be expressing SCGF in the stroma of ovarian cancer:

[0444] In EOC cryosections, the staining patterns are similar for SCGF and FAPa(Figure 11 A). However since all the antibodies were produced in mice, we were not able to conduct double staining experiments. Therefore, we used a virtual overlay technology to overlay the images from staining with CD68, SCGF and FAPa. These were 5pm thick consecutive cryosections stained with each marker in IHC. CD68 is a monocyte / macrophage marker. Due to challenges in cutting tissue sections that directly overlap with each other, it is difficult to make any conclusions from the overlay images. However there seems to be overlap between the SCGF and FAPa regions (Figure 1 IB). So FAPa and SCGF expressing (or producing) cells are in close spatial proximity.

[0445] These findings were validated with a double staining for SCGF and FAPa (rabbit antibody) and are shown in Figure 11C.

[0446] Treatment of EOC patient-derived tumour

[0447]

[0448] with the anti-SCGF antibody leads to T cell and activation in 75% of the cases

[0449] A cohort of 40 patient-derived tissues were cultured either with anti-SCGF antibody or left untreated. At the time of harvest, the tissues were each processed for multiplex cytokine measurement and for immunohistochemistry followed by whole slide imaging and semi-automated quantification of CD3+and CD8+cells. A T cell expansion was observed in 75% of tissues: these tissues exhibited significantly elevated T cell and CD8+cytotoxic T cell numbers per mm2after anti-SCGF (Figure 14). Additionally, we stained Granzyme B in a subcohort of tissues and in tissues where T cells and CD8+ cytotoxic T cells are expanded, the number of Granzyme T cells are also increased, showing that the expanded T cells are activated (Figure 14).

[0450] Treatment of EOC patient-derived tumour

[0451]

[0452] with anti-SCGF leads to the molecular si

[0453]

[0454] of a favourable immune

[0455]

[0456] In order to confirm the results depicted in Figure 7 showing modulation of various cytokines and chemokines in 2 patient-derived explant tissue cultures, we treated a series of n = 40 tissues and observed that systematically, tissues with a higher HGF concentration tend to resist to anti-SCGF antibodies and account for 25% of total tissues. In these non-responders, the same patterns are observed as in Ovar0047. In the rest of tissues, accounting for 75% of treated tissues, the tissues activate a signature of an anti-tumour immune response and respond in a similar fashion to Ovar0045, The results are depicted in Figure 13. Interestingly, the 25% non-responding tissues also harboured extremely elevated levels of SCGF at treatment onset, which allows hypothesizing that a higher antibody treatment might break resistance in that case.

[0457] The following modifications were observed: in 75% of treated tissues, anti-SCGF treatment leads to an increase in at least three of the following factors: IL-8, IL-6, GM-CSF, GROa, IL-lb, M-CSF, MIP-la, MIP-lb, all indicative of macrophage repolarization in situ (Figure 15A);

[0458] in 75% of treated tissues, anti-SCGF treatment leads to an increase in at least three of the following factors: IFN-a2, IFN-g, IL-12p70, TNF-a, IL-2, CXCL9, CXCL10, indicative of an ongoing active Thl / cytotoxic anti-tumour immune response (Figure 15B);

[0459] in 75% of treated tissues, anti-SCGF treatment leads to the elevation of IL-16, IL-18 and CCL27, three factors involved in T cell survival and recruitment (Figure 15C).

[0460] These results are presented in a simplified version for clarity using a score for macrophage repolarization and a score for Thl / cytotoxic T cell activation. The method to generate the scores is described in Figure 15E. A response specific to one patient is exemplified in Figure 15A-B.

[0461] The molecular signature to anti-SCGF response in

[0462]

[0463] mirrors the

[0464]

[0465] of TAMs, in which anti-SCGF activates an anti-viral

[0466]

[0467] In TAMs isolated from malignant ascites, the molecular response signature to anti-SCGF treatment can be reproduced (Figure 15 F), suggesting that a proportion of the effects observed in tissues come from the repolarized macrophages.

[0468] Additionally, the TAMs treated with anti-SCGF also exhibit a secretory signature typical of the activation of an antiviral response program (including an increase in various interferons and in members of the IL- 10 superfamily) (Figure 15 F).

[0469] to anti-SCGF antibody originates from the neutralization of SCGF and not from any Fc- innate immune cell activation.

[0470] The effects observed after anti-SCGF treatment are specifically due to its Fab-mediated binding. This is supported by the following data:

[0471] (i) A cohort of 4 patient-derived tissues were treated with the anti-SCGF antibody or with an antibody of the same structure and produced in the same batch, that recognizes an irrelevant (viral) antigen, known to be absent from the tissues. While the anti-SCGF antibody triggered specific effects, there was no distinction between tissues left untreated and tissues treated with the irrelevant antibody (Figure 16 A)

[0472] (ii) Should an Fc-dependent effect be triggered by the anti-SCGF antibody, it would naturally affect all monocytes and monocyte-derived macrophages, which all express Fc receptors abundantly. This was excluded in a cell culture experiment (Figure 16B). The molecular changes in secretions are exclusively observed tumour-associated macrophages. Anti-SCGF does not trigger any modification in monocytes or monocyte- derived macrophages.

[0473] Treatment of EOC patient-derived tumour explants with the anti-SCGF antibody increases apoptotic tumour cell death

[0474] To test whether anti-SCGF treatment leads to tumour cell death in the tumour microenvironment, a series of anti-SCGF treated explants were submitted to a stain for activated caspase 3, which demonstrated that the anti-SCGF treatment is associated with increased apoptotic cell death. To specifically demonstrate tumour cell death, the tissues were submitted to an EpC AM-activated caspase 3 double immunohistochemistry. The proportion of apoptotic tumour cells is defined as the ratio between the double positive cells and the EpCAM positive cells. Semiautomated analysis of the stain revealed an increased proportion of apoptotic tumour cells after anti-SCGF treatment both in EOC and in one CUP (Figure 17A-B). In one stomach cancer explant culture, the expression of EpCAM by cancer cells was lost, making the double immunostaining non-contributive. Therefore, we analysed the single activate caspase 3 immunostaining, and restricted the analysis to tumour epithelial islets, which were automatically detected by a miniNET Al classifier in Halo. In stomach cancer as well, anti-SCGF treatment leads to increased cell death in epithelial islets (Figure 17C).

[0475] Treatment of EOC patient-derived tumour explants with the anti-SCGF antibody reorganize the collagen fibre architecture around the tumour and facilitates T cell migration

[0476] In EOC tissue explants treated with anti-SCGF, one can observe that the collagen fibres (shown in blue in a Masson trichrome stain, Figure 18A) appear less dense and somewhat less aligned. This can be quantified thanks to second harmonics generation imaging, where 3D images are acquired at high resolution and the collagen fibres appear in white. The corresponding quantifications confirm that anti-SCGF treatment leads to slightly shorter fibres and more angles between the fibres (Figure 18C).

[0477] Consistently, we find that EOC specimens with high SCGF concentration exhibit a fibrotic component (Figure 18D) that can be quantified with second harmonics generation imaging (Figure 18E). Treatment of EOC patient-derived tumour explants with the anti-SCGF antibody leads to T cell expansion and fibre reorganization via the repolarization of tumour-associated macrophages in situ Because anti-SCGF treatment triggers a series of effects including macrophage repolarization, we analysed the causality between the different events triggered. For this purpose, a subcohort of n = 9 EOC explants were treated with clodronate liposomes (or PBS liposomes) prior anti-SCGF treatment. Clodronate liposomes are take up by macrophages and block all their functions. In these settings, one can see the effects of anti-SCGF treatment that are independent of macrophages. In the absence of functional TAMs, anti-SCGF treatment is not associated with fibre reorganization. The collagen fibres remain densely aligned (Figure 19 A) and most likely the tumour remains inpenetrable by T cells because of the barrier of collagen fibres. Accordingly, in the EOC explants pre-treated with clodronate liposomes, the numbers of T cells after anti-SCGF treatment are significantly lower (Figure 19 B).

[0478] Proposed mode-of-action of anti-SCGF treatment as a standalone immunotherapy in solid tumours. Based on all results mentioned above, we propose a mode-of-action of anti-SCGF treatment as a standalone immunotherapy in solid tumours (Figure 20).

[0479] (1) The first event is TAM repolarization in situ with the activation of an antiviral program; (2) Repolarized TAMs produce a series of cytokines and chemokines that support T cell expansion, chemotaxis and activation with production of granzyme B, leading to tumour cell death;

[0480] (3) Repolarized TAMs produce elevated amounts of TNFa, which could also be cytotoxic to tumour cells;

[0481] (4) Repolarized TAMs produce elevated amounts of enzymatically active matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9);

[0482] (5) In tumours burdened with a surrounding fibrotic component rendering them impenetrable by T cells, TAM-derived MMP-2 and MMP-9 (with maybe other factors) cleave collagen fibres and reorganize the fibre network into a more loosen network, compatible with T cell migration, further supporting invasion of the tumour by surrounding T cells;

[0483] (6) Tumours cells undergo apoptosis because of activated T cells and of TAM-derived TNF-a. When comparing anti-SCGF to other immunotherapies (see Figure 14 for a comparison with Nivolumab in EOC), the high response rate observed can be explained by the fact that the anti-SCGF antibody primarily tackles TAMs. While tumour are very heterogeneously infiltrated by T cells - which partly explains the failure of immune checkpoint blockade, at least in cold tumours -the infiltration and abundance of TAMs in solid tumours is universal.

[0484]

[0485] from other tumour to anti-SCGF treatment as a standalone i

[0486]

[0487] We evaluated the effects of anti-SCGF as a standalone immunotherapy in other solid tumour types and observed that anti-SCGF antibody systematically has an effect in all tumours tested so far, allowing to foresee a broad range of action. Indeed,

[0488] (1) SCGF levels are elevated in tissue lysates from other solid tumours including colorectal cancer (primary tumours and liver metastases), pancreatic cancer, stomach cancer, malignant melanoma, lung cancer, rare tumours such as CUP or MMMT, head and neck cancer. Similarly to EOC, anti-SCGF bind to stromal areas in these tumours (Figure 21). (2) Anti-SCGF treatment triggers a characteristic molecular signature as in EOC (Figure 15 D).

[0489] (3) Additionally to the molecular responses observed, we have observed increased T cell or CD8+ cytotoxic T cell numbers in these tissues after anti-SCGF treatment (Figure 21) (4) Importantly, we have been able to confirm that anti-SCGF treatment increases tumour cell killing, at least in CUP, stomach cancer in addition to EOC (Figure 21).

[0490] Possible between Anti-SCGF and

[0491]

[0492] In EOC samples from patients who have received chemotherapy, the concentration of SCGF is generally higher compared to the concentration in lysates from chemotherapy-naive patients (Figure 22A). Furthermore, the treatment of EOC samples from chemotherapy-naive patients with carboplatin in vitro leads to an increase in SCGF concentration (Figure 22B). The amplitude of this increase is directly dependent on the concentration of carboplatin added on the tissue (Figure 22C). Similar results were obtained with explant cultures of liver metastases treated with oxaliplatin (Figure 22D) or of pancreatic cancer treated with FOLFIRI (folate, fluorouracile and irinotecan) (Figure 22E). These results suggest that, while we observe response to anti-SCGF treatment in tissues independently of the chemostatus, clinically, patients might be even more responsive to anti-SCGF after having received chemotherapy. Synergy between anti-SCGF treatment and HGF receptor inhibition in explants from EOC

[0493] In our cohort of EOC tissues, the concentration of hepatocyte growth factor (HGF) is higher in tumours harbouring high SCGF concentration (cut-off at the median) (Figure 23 A) and most interestingly, EOC explants that do not respond to anti-SCGF have also slightly elevated HGF concentrations (Figure 23 B). We treated a subcohort of EOC explants with either anti-SCGF, or Capmatinib to inhibit HGF receptor c-Met, or a combination of both. EOC explants exhibited a synergistic induction of Thl / cytotoxic factors (Figure 23 C) and a synergistic expansion of CD8+cytotoxic T cells (Figure 23 D-E).

[0494] From this subcohort of 10 patient-derived cultures, the addition of Capmatinib appears to bring the response rate to anti-SCGF from to 100%.

[0495] Anti-SCGF synergizes with adoptive cell therapy (both autologous TILs and CAR T cells)

[0496] In light of our data demonstrating that anti-SGCF supports the expansion and activation of autologous T cells, but also their infiltration into the tumour (Figure 14), through versatile mechanisms (Figure 20), we evaluated whether this response would also apply to T cells that would be adoptively transferred. This hypothesis was tested with two independent experiments (Figure 24).

[0497] (i) One lung metastasis of a MMMT was cultured as explants with addition of autologous T cells from the patient. The autologous T cells had been isolated from the patient’s tumour and expanded in vitro. The autologous T cells were added on top of the explants, which were also treated with anti-SCGF. Anti-SCGF treatment maximized the number of total tumour- infiltrating CD8+ cytotoxic T cells after 24 hours of culture.

[0498] (ii) One primary colorectal cancer sample was cultured as explants with the addition of non-autologous CAR T cells. After 24 hours, the tissue was harvested and in this case, we could track the CAR T cells specifically thanks to their prior fluorescent labelling. The infiltration of the anti-SCGF-treated explants by CAR T cells was more than doubled compared to the untreated explant.

[0499] These results confirm that anti-SCGF-mediated support of TIL infiltration also applies to adoptively transferred T cells and in this case, as CAR T cell therapy has largely failed in solid tumours, might break the tumour resistance to T cell infiltration. Synergy between anti-SCGF and immune checkpoint inhibition (exemplified with Nivolumab) Responses to ICB in EOC are very durable but the response rate is very low (approximately 12%) and the resistance mechanisms are unclear so far. But because of these low response rates, ICB is not approved for the treatment of EOC so far. We tested and observed synergy between ICB (we used Nivolumab as anti-PD-1 antibody) and anti-SCGF in explants (Figure 25). In explants from EOC, melanoma or liver metastases, the concentration of SCGF is increased after treatment with Nivolumab (Figure 25 A-C). Furthermore, in EOC explants treated with both Nivolumab and anti-SCGF, we observed one case of synergistic proliferation of CD8+ cytotoxic T cells (Figure 26 D). Additionally, we observed two cases of synergistic induction of apoptotic tumour cell death (Figure 26 E) that seemed independent from T cell numbers (Figure 26 F).

[0500] Synergy between anti-SCGF and CCR5 inhibition (exemplified by maraviroc)

[0501] In some tissue explants, we observed that SCGF levels increase upon maraviroc-mediated CCR5 inhibition and that CCL5 levels increase upon anti-SCGF treatment. We hypothesized that there could be a mechanism of compensation involving SCGF and CCL5 and tested the effects of a double treatment. Interestingly, in one EOC sample that does not respond to anti-SCGF, we observed synergistic response at the molecular level (TAM repolarization and the markers of the activation of a Thl / cytotoxic immune response were consistently elevated, exclusively after the double treatment) (Figure 26 A) and at the cellular level (CD8+ cytotoxic T cell numbers were elevated after double treatment, but not after anti-SCGF or after maraviroc treatment alone) (Figure 26 B). We observed similar findings in explants from a Kruckenberg tumour (a stomach cancer growing in the ovaries) (Figure 26 C), suggesting a synergy between the neutralization of SCGF and CCL5.

[0502] SCGF acts as a growth and survival factor in cancer cells.

[0503] In a series of cancer cells (primary cells and cell lines), we were able to show that recombinant human SCGF acts as a growth and survival factor (Figure 27). This helps us envision that neutralization of SCGF might trigger cancer cell death.

[0504] SCGF is associated with CREB phosphorylation in cancer.

[0505] We treated TAMs with anti-SCGF and analysed the modulated signalling pathways using multiplex analysis of protein phosphorylation with the Luminex technology and found that anti-SCGF treatment of TAMs decreases phosphorylation of the transcription factor CREB (Figure 28).

[0506] In whole tumour lysates, SCGF high samples exhibit a lower phosphorylation of CREB and c-Jun (Figure 28).

[0507] Concepts and evidence behind invention

[0508] Ovarian cancer is the deadliest gynaecological malignancy and new treatment options are required to improve survival rates (Bowtell et al, 2015). SCGF is a largely uncharacterized hematopoietic mediator but has recently been described in different cancer entities as being associated with a more aggressive cancer type. Mechanistically, SCGF might promote inflammation and cancer stem cell (CSC) proliferation as reported in murine and in vitro models (Da Riva et al, 2011; Levina et al, 2008). In this project, we have identified SCGF as a novel therapeutic target for the immunotherapy or solid tumours based on its high concentration in tumour lysates. We produced chimeric anti-SCGF antibodies using sequencing data obtained from a unique murine hybridoma producing a mouse anti-SCGF antibody. Three different antibodies were cloned and purified; two scFv fragments ligated to mouse IgG2a and human IgGl and one full length anti-SCGF antibody ligated to human IgGl (heavy chain) and human Ig kappa (light chain). The scFv antibodies are aglycan but the full length antibody can be glycosylated at Asn297 and facilitates effector immune cell binding. There are advantages and disadvantages of both designs but by comparing them, we determined the best approach of targeting SCGF. The binding and function of the chimeric antibodies are comparable to the hybridoma supernatant containing murine anti-SCGF antibody.

[0509] We were able to successfully produce all three antibodies and confirm their size and binding capacity. When the antibodies were run on SDS Page gels, there was a clear separation between the reducing and non-reducing conditions and the bands were present at the expected locations. The full length antibody was more difficult to produce due to the double transfection with heavy and light chain plasmids. This antibody was finally produced in both CHO-S (transient transfection) and HEK (stable transfection) cells. We had to transfect twice the number of cells and the efficiency of the transfection was low when compared to the scFv antibodies, which could be produced with a high yield. We purified the antibody from both cell types and all constructs passed the SDS-Page quality control (Figure 4).

[0510] We confirmed the binding ability of our produced antibodies by capture ELISA and determined that the binding side is precisely on the C-lectin-type domain (Figure 5). We tested the scFv-mIgG2a anti-SCGF antibody and the mouse hybridoma supernatant in immunohistochemistry on consecutive cryosections from untreated ovarian cancer tissues and a similar staining pattern could be obtained (Figure 6).

[0511] The scFv-hlgGl anti-SCGF antibody was tested in explant tissue culture from a large cohort of patient-derived tumours from epithelial ovarian cancers (n = 40) and on several tissues from colorectal cancer-liver metastases, pancreatic cancer, stomach cancer, melanoma, as well as on some rare tumours. We identified a series of consistent changes in the molecular landscape in whole tissue lysates upon treatment with anti-SCGF. In the EOC cohort, these changes, indicated as response, were present in 75 % of the treated tissues and included an increase in a series of factors indicative of TAM repolarization, a series of factors indicative of Thl / cytotoxic immune response activation and a series of factors involved in T cell recruitment (Figures 13, 15).

[0512] Functionally, these changes were accompanied by the proliferation and expansion of T cells and CD8+cytotoxic T cells that were activated (Granzyme B+) and therefore capable of killing tumour cells. Increased apoptotic tumour cell death was also induced by SCGF neutralization after 48 hours of treatment.

[0513] Response to immunotherapy but also to radiotherapy and chemotherapy relies on a strong infiltration of the tumour by effector T cells (Binder et al. 2015, Lee et al. 2009, Zitvogel et al.

[0514] 2010). Therefore, these results strongly indicate that anti-SCGF treatment can break resistance to treatment in many indications.

[0515] In terms of mode-of-action, we have confirmed that the effects triggered by anti-CSGF antibodies are Fab-dependent (Figure 16), as speculated (since the scFv-hlgGl is aglycan due to the N297Q mutation).

[0516] The cytokines affected by SCGF inhibition in ovarian cancer tissue culture can be grouped into different categories based on their functions. SCGF inhibition results in an increase in pro-inflammatory, Thl supportive cytokines that promote the activation of an anti-tumour adaptive immune response. Also, an increase in cytotoxic factors is observed and finally, anti-SCGF triggers an increase in a multitude of chemokines that boost intra-tumour infiltration by immune cells (CXCL9, CXCL10, IL- 16, IL- 18, CCL27) as well as factors supportive of T cell survival (such as IL-9). CCL27 is an indispensable factor in T cell chemoattraction to tumour sites (Pivarcsi et al.

[0517] 2007). IL- 16 recruits and activates Thl CD4+ T cells and IL- 18 synergizes with other pro-inflammatory cytokines to promote IFN-g production (Zhou et al. 2020). In some tissues, a decrease in pro-angiogenic factors can also be observed. These include VEGF, PDGF, FGFb (Cojoc et al, 2013; Demir etal, 2016). 1

[0518] Based on the observation that SCGF inhibition in explant tissue culture led to modulation of the expression of soluble factors regulating angiogenesis, macrophage activation and T cell chemotaxis and activation, we hypothesized that SCGF might be involved in these processes. We therefore compared the expression of SCGF (as measured in Luminex) to CD3, CD8, CD163 and CD 105 expression (measured by immunohistochemistry). CD 105 is a marker for immature blood vessels, CD3 and CD8 are T-cell markers and CD163 is a marker for tumour-associated macrophages. There was no correlation between SCGF and CD 105 or CD8 (Figure 8A, 8B). Therefore, SCGF might control angiogenesis in collaboration with other factors. There were two sets of tissues when SCGF was compared to CD3 and CD163 expression (Figure 8C, 8D). There were some tissues with low SCGF and low CD3, CD163 and others with low SCGF but high CD3, CD163. Once again, it appears that SCGF alone does not control CD3+T-cell or CD163+macrophage infiltration in ovarian cancer.

[0519] SCGF does not directly promote angiogenesis or immune cell infiltration but it still contributes to other pro-tumour functions. One open question is what cell type secretes SCGF in the tumour microenvironment. Based on a literature analysis and data from our laboratory, we hypothesized that macrophages and FAP+cancer associated fibroblasts secrete SCGF in ovarian cancer. The mRNA for SCGF is found in myeloid cells, restricted to immature neutrophils and it is expressed in the monocytic THP-1 cell line (Hiraoka et al, 1997; Perrin et al, 2001). Resident lung macrophages secrete SCGF that recruit epithelial progenitor cells in cystic fibrosis (Gilpin et al, 2013). In our laboratory, macrophages from breast or ovarian cancer ascites were positive for SCGF. There was a difference in intensity of SCGF in the cells indicating that the levels vary from cell to cell (Figure 9). The SCGF was localized in the plasma membrane. This localization is not observed in tissue sections which could be due to the fact that pleural effusions are detached macrophages and therefore these cells with their enormous plasticity could be in a different state of activation or migration. Cancers are known to have both Ml and M2 macrophages in the tumour microenvironment. Gonzalez-Dominguez et al. proposed CLEC5A and CD 163 as markers to distinguish between Ml and M2 macrophages in melanoma. The CLEC5A+cells are the Ml -like inflammatory cells and the CD163+cells are the resident M2-like anti-inflammatory cells (Gonzalez-Dominguez et al, 2015). We established double immunofluorescent staining with CLEC5A and SCGF and CD 163 and SCGF on the macrophages from the pleural effusions. In both sets of staining, there were double positive cells indicating that SCGF is produced by macrophages irrespective of polarization status (Figure 10A, 10B). Immunohistochemistry analysis with anti-SCGF antibody revealed staining in cells with a fibroblastic morphology. Consecutive sections stained with FAPa revealed a similar staining pattern. These data suggesting that SCGF might be produced by cancer-associated fibroblasts are pertinently supported by several published reports. Indeed, SCGF mRNA is detectable in myeloid cells and fibroblastic cell lines in vitro (Hiraoka et al, 1997). In Gastrointestinal stromal tumours, SCGF protein was detected in the stroma by western blot and immunohistochemistry (Da Riva et al, 2011). We were not able to establish double staining because all the antibodies available at the laboratory were anti-mouse primary antibodies. Therefore we stained consecutive 5pm cryo-sections of direct untreated ovarian cancer tissue with FAPa, SCGF and CD68 (macrophage / monocyte marker). In collaboration with Nektarious Valous (NCT Heidelberg), we were able to generate virtual overlay images to check if there is a co-expression of SCGF and FAPa or SCGF and CD68. We validated these data thanks to the use of another anti-FAPa antibody produced in rabbits and the co-expression of SCGF and FAPa by CAFs is established. The works published by Weiland et al. corroborate our data as they illustrate that CAFs regulate CSC phenotypes by providing regulatory factors such as HGF and IL-6. In pancreatic ductal carcinoma, immunosuppression is mediated by CXCL12 secreted by CAFs. The tumours do not respond to immune checkpoint inhibitors until CXCL12 is also inhibited (Feig et al, 2013). These data support our hypothesis linking SCGF expression and CAFs. Indeed, these cytokines are decreased in our Luminex analysis after anti-SCGF treatment in ovarian cancer explant tissue cultures (Figure 7).

[0520] Thanks to sequential in situ hybridization-immunohistochemistry experiments, we tracked the mRNA expression of the cleclla gene and combined with surface markers of CAFs and macrophages. The results further confirmed that CAFs produce SCGF as we see cleclla mRNA in FAPalpha+cells. However, this is not the only cellular source of SCGF in the tumour stroma, as we also observe cleclla mRNA in CD14+ and CD68+ macrophages. Altogether our data point at a mixed, fibroblastic-monocytic cellular origin of SCGF in the cancer stroma.

[0521] The cytokine data from our experiments show that blocking SCGF produces some important anti-tumour effects. Levina et al. described that protein lysates from lung cancer stem cells had high SCGF, SCF and CXCL12 levels associated with the stem cell phenotype. These cells were chemotherapy resistant in-vitro and demonstrated high tumorigenic and metastatic potential in-vivo. Inhibiting SCGF can decrease the levels of all these cytokines thus reducing overall sternness. This could prevent the acquisition of chemotherapy resistance and relapse. These are major clinical challenges in ovarian cancer and currently there are no treatments for chemotherapy resistant ovarian cancer. Further analysis is needed to transition these antibodies from the laboratory to the clinic but anti-SCGF treatment could be a promising addition to current ovarian cancer therapy.

[0522] The original mode-of-action of anti-SCGF, where the TAMs are the primary target of modulation, probably explains the extremely high response rates that we have obtained with this drug compared to what is achieved with standard immunotherapy (75% response to anti-SCGF in contrast to 12% response to Nivolumab). Indeed, while T cell infiltration is heterogeneous in solid tumours, TAM abundance is a constant. For this reason, anti-SCGF breaks resistance to therapies that fail because of poor tumour infiltration by TILs, like adoptive T cell transfer, CAR T cell therapy (Sterner and Sterner, 2021), on top of standard chemotherapy, which also relies on T cell infiltration for the mounting of a long-term response (Binder et al. 2015).

[0523] During the study of the mode-of-action of anti-SCGF antibodies in EOC, we demonstrated that anti-SCGF leads to the reorganization of tumour-associated collagen fibres that sometimes surround tumours. Anti-SCGF trigger the production of active enzymes by TAMs that in turn act on the dense fibre network - typically associated with little or no TIL in the tumour or with a “tumour- excluded” infiltration pattern where TILs accumulate at the invasive margin but fail to infiltrate the tumour core. The anti-SCGF-modulated network is composed of loosen fibres that are less aligned and that are compatible with infiltration by immune cells (Figures 18-20).

[0524] Anti-SCG monoclonal antibody of the invention as compared to prior art Anti-SCGF monoclonal antibodies

[0525] Provided herein is an innovative approach for immunotherapy of solid tumors using the novel neutralizing anti-SCGF / CLEClla monoclonal antibody of the invention. SCGF proteins are abundant in the stroma of various solid tumors and their levels rise with tumor progression, indicating chemotherapy failure. In addition, elevated SCGF levels are predictive of resistance to immunotherapy, as demonstrated in a prospective study. Finally, SCGF neutralization in patient-derived tumor explants (PDEs) results in a significant reorganization of the tumor microenvironment that ultimately enhances the anti-tumor immune response. The observed effects include the reprogramming of TAMs in situ, the remodeling of the dense collagen fiber network surrounding tumors, enhanced infiltration of the tumor by tumor-associated cytotoxic T cells, activation of these T cells, and apoptotic death of tumor cells. The therapeutic anti-SCGF antibody is currently a chimeric single chain fraction variable (scFv) coupled to a Fc-silent human IgGl (also termed “8326.1” herein and used in the experiments as described below). The scFv part selectively binds the functional C-lectin type domain of SCGF involved in protein-protein interaction. To validate the functionality of 8326.1 in comparison with different commercially available anti-SCGF antibodies, we have developed two robust in vitro assays using the human monocytic cell lines THP-1 (DSMZ number ACC 16) and MonoMac6 (DSMZ number ACC 124). Specifically, we tested described molecular functions of SCGF in inhibiting cellular activation and facilitating cellular growth. Accordingly, we expected an induction of cellular activation as well as inhibition of proliferation when providing active anti-SCGF antibodies. Our previous results show that SCGF-neutralization exerts an activating effect on macrophages, enhancing pro-inflammatory cytokines like tumor necrosis factor a (TNFa) or Interleukin (IL)-13 in tumor explants. Accordingly, we tested if anti-SCGF antibodies might have an activating effect on differentiated human THP-1 cells, enhancing pro- inflammatory cytokine secretion after stimulation. Our results show that differentiated THP-1 cells can be stimulated by the bacterial cell wall component lipopolysaccharide (LPS) to secrete TNFa and IL-113, and this secretion was enhanced by up to 25% by the addition of the antibody 8326.1 in a dose dependent fashion (Figure 29B, C). A similar albeit less pronounced effect was observed for the commercially available antibody obtained from Proteintech (60-295 -I-Ig), while the antibody obtained from R& D (MAB1904) showed only minor effects in this assay. In different experiments using the same experimental setup we tested the effect of anti-SCGF antibodies on IL-113 secretion. Here, we observed a strong induction of LPS-induced IL- 113 secretion when the antibody 8326.1 was present (Figure 29D). While addition of the commercially available antibodies showed also an enhancing effect in terms of IL- 113 secretion, the antibody 8326.1 proved to be superior in inducing this cytokine (Figure 29D).

[0526] Taken together, our data clearly show a differential effect of the 3 antibodies examined: While the antibody 8326.1 showed a high, dose dependent activity, the antibody obtained from Proteintech showed a variable, medium activity and the antibody obtained from R& Dshowed a neglectable effect on the secretion of TNFa. It is most likely that different antibodies, which were raised in different animals in response to different vaccination regimes, bind to different epitopes on the molecule SCGF. Thus, the differential activities as observed in our assays may likely result from different binding sites on SCGF, resulting in different modes of action with respect to SCGF binding to its receptor on cells. This is especially true in light of several putative receptors that may bind to and mediate the effects of SCGF (see e.g. Wang M, et al. Molecular structure, expression, and functional role of Cleclla in skeletal biology and cancers. J Cell Physiol. 2020; 235: 6357-6365). Items of the invention

[0527] The present invention also pertains to the following items.

[0528] 1. An antigen binding protein that specifically binds to Stem Cell Growth Factor (SCGF), wherein said antigen binding protein competes for binding to SCGF with a reference antibody comprising a heavy chain variable region of the amino acid sequence in SEQ ID NO: 1; and a light chain variable region of the amino acid sequence in SEQ ID NO: 2.

[0529] 2. An antigen binding protein that specifically binds to SCGF, wherein said antigen binding protein comprises either:

[0530] (i) a combination of a light chain variable domain and a heavy chain variable domain selected from the group of combinations consisting of:

[0531] a heavy chain variable domain having a sequence with at least 90% identity to SEQ ID NO: 1; and

[0532] a light chain variable domain having a sequence with at least 90% identity to SEQ ID NO: 2; or

[0533] (ii) a complementarity determining region 3 of the heavy chain (CDRH3) comprising or consisting of the amino acid sequence of SEQ ID NO: 5 and a complementarity determining region 3 of the light chain (CDRL3) comprising or consisting of the amino acid sequence of SEQ ID NO: 8.

[0534] 3. The antigen binding protein of item 2, further comprising one or more CDRs selected from the group consisting of a CDRH1 comprising or consisting of the amino acid sequence of SEQ ID NO: 3, a CDRH2 comprising or consisting of the amino acid sequence of SEQ ID NO: 4, CDRL1 comprising or consisting of the amino acid sequence of SEQ ID NO: 6 and a CDRL2 comprising or consisting of the amino acid sequence of SEQ ID NO: 7.

[0535] 4. An antigen binding protein that specifically binds to Stem Cell Growth Factor (SCGF), wherein said antigen binding protein.

[0536] comprises a CDRH1 comprising or consisting of the amino acid sequence of SEQ ID NO: 3, a CDRH2 comprising or consisting of the amino acid sequence of SEQ ID NO: 4, a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 5, a CDRL1 comprising or consisting of the amino acid sequence of SEQ ID NO: 6, a CDRL2 comprising or consisting of the amino acid sequence of SEQ ID NO: 7, and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 8; and / or

[0537] binds to an epitope of SCGF that comprises or consists of the amino acid sequence PVWLGVHD (SEQ ID NO: 11) of SEQ ID NO: 9.

[0538] The antigen binding protein of item 4, wherein the antigen binding protein comprises a heavy chain variable domain having a sequence with at least 90% identity to SEQ ID NO: 1; and / or a light chain variable domain having a sequence with at least 90% identity to SEQ ID NO: 2.

[0539] The antigen binding protein of any one of the preceding items, wherein the antigen binding protein comprises an antibody, a SCGF-binding fragment of an antibody or an antibody-like protein.

[0540] The antigen binding protein of item 4, wherein:

[0541] (i) the antibody is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, or a rodent, in particular a mouse antibody or from any other species; and / or

[0542] (ii) the antibody is a mono-specific, bi-specific, tri-specific, or multi-specific antibody; and / or (iii) the antibody is a single chain antibody, a single chain variable fragment antibody, a diabody, a Fab fragment, or an F(ab)2 fragment; and / or

[0543] (iv) the antibody is selected from an IgA, IgE, IgG, in particular IgGl, an IgG2 antibody, an IgG3 antibody, and an IgG4 antibody; and IgM; or

[0544] (v) the antibody-like protein is selected from the group consisting of lipoprotein-associated coagulation inhibitor (LACI-D1); affilin, e.g. human-γ B crystalline or human ubiquitin; cystatin; Sac7D from Sulfolobus acidocaldarius; lipocalin, anticalin; designed ankyrin repeat domains (DARPins); SH3 domain of Fyn; Kunits domain of a protease inhibitor; monobodies, e.g. the 10thtype III domain of fibronectin; adnectins: cysteine knot miniproteins knottins; atrimers; evibodies, e.g., affibodies, e.g. three-helix bundle from Z- domain of protein A from Staphylococcus aureus Trans-bodies, e.g. human transferrin; tetranectins, e.g. monomeric or trimeric human C-type lectin domain; microbodies, e.g. trypsin-inhibitor-II; an armadillo repeat protein; tetraspanins, B-type lection domain containing elements, EZH type domain containing elements, avimers, nanofitins and affilins.

[0545] 8. The antigen binding protein of any one of the preceding items, wherein the antigen binding protein has at least one of the following characteristics:

[0546] a. binds to SCGF with a Kdof less than 1 nM, of less than 100 pM, of less than 10 pM, or of less than 5 pM; and / or

[0547] b. blocks the binding of SCGF to a cell responsive to SCGF with an IC50of less than 1 nM, preferably less than 200 pM; and / or

[0548] c. decreases secretion of pro-angiogenic cytokines, sternness-related cytokines, immunosuppressive molecules and / or inflammatory cytokines and / or chemokines.

[0549] 9. The antigen binding protein of any of items 1 to 8, further comprising at least one:

[0550] (i) a second antigen binding protein specifically binding to a target different from SCGF, preferably to the ectodomain of a different protein than SCGF, wherein the target different from SCGF is preferably an immune checkpoint protein (such as PD1, PDL- 1 or CTLA-4), CCR5, HGF receptor, or an IGF-1 receptor;

[0551] (ii) a second antigen binding protein specifically binding to an SCGF receptor;

[0552] (iii) a toxin, and / or

[0553] (iv) a label, preferably a radionuclide or a fluorophore;

[0554] (v) a polypeptide comprising or consisting of a transmembrane domain and, optionally an endo domain;

[0555] (vi) a drug,

[0556] (vii) a chemokine,

[0557] (viii) a cytokine,

[0558] (ix) an enzyme,

[0559] (x) a component modulating serum half-life, and / or

[0560] (xi) an Fc part of an antibody.

[0561] 10. The antigen binding protein of item 9, wherein:

[0562] (i) the target different from SCGF is selected from the group consisting of C-Lectin-Type domains, immunomodulatory or homeostasis-regulating domains different from the respective domains of SCGF; and / or (ii) the transmembrane domain is the transmembrane domain of CD28 and in tandem the intracellular signalling transducing domains of CD3-zeta and CD28.

[0563] The antigen binding protein of item 10, wherein the other or similar C-Lectin-Type domains or immunomodulatory or homeostasis-regulating domains are selected from the group consisting of Stem Cell Factor (SCF), GM-CSF, interleukin-6, 0X40, CD40, CD40L, LAG3, TIM-3, and fibroblast activating protein 1 (FAP alpha).

[0564] The antigen binding protein according to item 10 alternative (i), wherein the target different from SCGF includes binding to proteins from the group consisting of Dectin- 1 and / or Dectin-2 or the group of mannose receptors (i.e. able to bind branched sugars with terminal mannose, fucose or N-acetyl-glucosamine).

[0565] The antigen binding protein of any one of the preceding items, wherein the antigen binding protein comprises an scFv fragment being fused to an Fc part, wherein the Fc part is preferably a human IgGl or mouse IgG2 Fc part and / or a genetically engineered Fc part that abrogates binding of Fc receptors.

[0566] A nucleic acid or a set of two nucleic acid molecules encoding the antigen binding protein of any one of the preceding items.

[0567] A recombinant expression vector or a set of two recombinant expression vectors comprising a nucleic acid molecule(s) according to item 11.

[0568] A host cell comprising the vector or set of two vectors of item 12.

[0569] A method of making the antigen binding protein of any one of items 1-10, comprising the step of preparing said antigen binding protein from a host cell expressing said antigen binding protein.

[0570] A pharmaceutical composition comprising at least one antigen binding protein according to any one of items 1 to 14, the nucleic acid according to item 14, or the vector according to item 15 and optionally further comprising a pharmaceutically acceptable excipient. The pharmaceutical composition of item 18 further comprising one or more compounds being selected from

[0571] (v) an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is preferably an antibody or a small molecule against PD1, PDL-1 or CTLA-4 and is most preferably Nivolumab,

[0572] (vi) an inhibitor of the chemokine receptor CCR5, wherein the CCR5 inhibitor is preferably Maraviroc,

[0573] (vii) anti-tumor lymphocytes, wherein the anti-tumor lymphocytes are preferably chimeric antigen receptor T-cells (CAR T-cells), T-cell-receptor-engineered T-cells (TCR T- cells), chimeric antigen receptor NK-cells (CAR NK-cells), NK cell receptor- engineered NK cells (NCR NK-cells), TCR / CAR hybrid T-cells, NCR / CAR hybrid NK-cells or tumor-infiltrating lymphocytes (TILs), and

[0574] (viii) a tyrosine kinase inhibitor, wherein the tyrosine kinase inhibitor is preferably an inhibitor of the HGF receptor and is most preferably Capmatinib.

[0575] A kit for the treatment of cancer, inflammation, or trauma comprising at least one antigen binding protein according to any one of items 1 to 13, the nucleic acid according to item 14, the vector according to item 15, or the host cell of item 16, the pharmaceutical composition of item 18 or 19, or a combination thereof.

[0576] A SCGF antagonist for use in treating or preventing a condition associated with cancer, inflammation and / or trauma in a subject.

[0577] The SCGF antagonist for use of item 21, wherein the SCGF antagonist is selected from the group consisting of:

[0578] (i) antibodies and SCGF-binding fragments thereof,

[0579] (ii) antibody-like proteins,

[0580] (iii) inhibitory variants of SCGF;

[0581] (iv) inhibitors of SCGF-activity;

[0582] (v) a nucleic acid encoding (i) to (iii); and

[0583] (vi) a vector comprising (v). 23. The SCGF antagonist for use of item 21, wherein the inhibitor of SCGF-activity is selected from the group consisting of:

[0584] (i) an oligonucleotide that specifically binds to SCGF, preferably a DNA-aptamer, D-RNA aptamer, or a L-RNA aptamer; or

[0585] (ii) an oligonucleotide selected from the group consisting of antisense DNA, antisense RNA, siRNA, and miRNA.

[0586] 24. The SCGF antagonist for use according to any of items 21 to 23, wherein the SCGF antagonist is the antigen binding protein of any one of items 1 to 13, the nucleic acid according to item 14, the vector according to item 15, the host cell according claim 16, the pharmaceutical composition of item 18 or 19, or a combination thereof.

[0587] 25. The SCGF antagonist for use according to any of items 21 to 24, wherein the cancer is resistant to standard therapy, preferably, wherein the cancer is resistant to chemotherapy and / or immunotherapy and / or radiation therapy.

[0588] 26. The SCGF antagonist for use according to any of items 21 to 25, wherein the cancer is a solid tumour.

[0589] 27. The SCGF antagonist for use according to any of items 12 to 25, wherein

[0590] (i) the cancer is selected from ovarian cancer, colorectal cancer, pancreatic cancer, lung cancer, renal cancer, bladder cancer, prostate cancer, breast cancer and skin cancer, preferably ovarian cancer or colorectal cancer;

[0591] (ii) the inflammation is selected from pulmonary fibrosis, multiple sclerosis, auto-immune diseases (lupus erythematosus, lichen planus, rheumatoid arthritis, Sjogren syndrome, Morbus Bechterew, primary sclerosing cholangitis, ulcerative colitis, Morbus Crohn, mesangioproliferative nephritis, Guillain-Barre-Syndrome, rheumatic fever, Hashimoto thyreoiditis and Morbus Still, Morbus Basedow, Diabetes mellitus type 1, Myasthenia gravis, idiopathic thrombocytopenia, celiac disease and sarcoidosis; and / or

[0592] (iii) the trauma can be of any mechanical, physical or chemical trauma to tissues or organs (e.g. traffic accident, stroke, cardiac infarction, pulmonary embolism).

[0593] 28. At least one antigen binding protein according to any one of items 1 to 13, the nucleic acid according to item 14, the vector according to item 15, or the host cell of item 16, the pharmaceutical composition of item 18 or 19, or a combination thereof and optionally in addition one of the further compounds being selected from items (i) to (iv) of item 19 for use in treating or preventing a disease, wherein the disease is preferably a cancer, an inflammatory disease and / or a trauma in a subject, wherein the subject is preferably an adult.

[0594] The at least one antigen binding protein, the nucleic acid according, the vector, the host cell, the pharmaceutical composition, or the combination thereof for use according to item 28, wherein the cancer is resistant to standard therapy, preferably, wherein the cancer is resistant to chemotherapy and / or immunotherapy and / or radiation therapy.

[0595] The at least one antigen binding protein, the nucleic acid according, the vector, the host cell, the pharmaceutical composition, or the combination thereof for use according to item 28 or 20, wherein the at least one antigen binding protein, the nucleic acid according, the vector, the host cell, the pharmaceutical composition, or the combination is used to treat cancer after the cancer has been treated by chemotherapy.

[0596] The at least one antigen binding protein, the nucleic acid according, the vector, the host cell, the pharmaceutical composition, or the combination thereof for use according to any of items 28 to 30, wherein the cancer is a solid tumour.

[0597] The at least one antigen binding protein, the nucleic acid according, the vector, the host cell, the pharmaceutical composition, or the combination thereof for use according to any one of items 28 to 30, wherein

[0598] (i) the cancer is selected from ovarian cancer, colorectal cancer, pancreatic cancer, lung cancer, renal cancer, bladder cancer, prostate cancer, breast cancer, leukemia, myeloma and skin cancer, preferably ovarian cancer or colorectal cancer;

[0599] (ii) the inflammatory disease is selected from pulmonary fibrosis, multiple sclerosis, autoimmune diseases (lupus erythematosus, lichen planus, rheumatoid arthritis, Sjogren syndrome, Morbus Bechterew, primary sclerosing cholangitis, ulcerative colitis, Morbus Crohn, mesangioproliferative nephritis, Guillain-Barre-Syndrome, rheumatic fever, Hashimoto thyreoiditis and Morbus Still, Morbus Basedow, Diabetes mellitus type 1, Myasthenia gravis, idiopathic thrombocytopenia, celiac disease and sarcoidosis; and / or (iii) the trauma is a mechanical, physical or chemical trauma to tissues or organs (e.g. traffic accident, stroke, cardiac infarction, pulmonary embolism).

[0600] The at least one antigen binding protein, the nucleic acid according, the vector, the host cell, the pharmaceutical composition, or the combination thereof for use according to any one of items 28 to 30, wherein the cancer is a hematologic malignancy. References

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Claims

1. CLAIMS1. An antigen binding protein that specifically binds to Stem Cell Growth Factor (SCGF), wherein said antigen binding protein3.(i) comprises a CDRH1 comprising or consisting of the amino acid sequence of SEQ ID NO: 3, a CDRH2 comprising or consisting of the amino acid sequence of SEQ ID NO: 4, a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 5, a CDRL1 comprising or consisting of the amino acid sequence of SEQ ID NO: 6, a CDRL2 comprising or consisting of the amino acid sequence of SEQ ID NO: 7, and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 8; and / or (ii) binds to an epitope of SCGF that comprises or consists of the amino acid sequence PVWLGVHD (SEQ ID NO: 11) of SEQ ID NO: 9.

2. The antigen binding protein of claim 1, wherein the antigen binding protein comprises a heavy chain variable domain having a sequence with at least 90% identity to SEQ ID NO: 1; and / or a light chain variable domain having a sequence with at least 90% identity to SEQ ID NO: 2.

3. The antigen binding protein of claim 1 or 2, wherein the antigen binding protein comprises or is an antibody, a SCGF-binding fragment of an antibody or an antibody-like protein.

4. The antigen binding protein of claim 3, wherein:7.(i) the antibody is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, or a rodent, in particular a mouse antibody or from any other species; and / or8.(ii) the antibody is a mono-specific, bi-specific, tri-specific, or multi-specific antibody; and / or (ii) the antibody is a single chain antibody, a single chain variable fragment antibody, a diabody, a Fab fragment, or an F(ab)2 fragment, single-chain antibody (VH-only antibodies) or a nanobody; and / or9.(iv) the antibody is selected from an IgA, IgE, IgG, in particular IgGl, an IgG2 antibody, an IgG3 antibody, and an IgG4 antibody; and IgM; or10.(v) the antibody-like protein is selected from the group consisting of lipoprotein-associated coagulation inhibitor (LACI-D1); affilin, e.g. human-γ B crystalline or human ubiquitin; cystatin; Sac7D from Sulfolobus acidocaldarius lipocalin, anticalin; designed ankyrin repeat domains (DARPins); SH3 domain of Fyn; Kunits domain of a protease inhibitor; monobodies, e.g. the 10thtype III domain of fibronectin; adnectins: cysteine knot miniproteins knottins; atrimers; evibodies, e.g., affibodies, e.g. three-helix bundle from Z- domain of protein A from Staphylococcus aureus Trans-bodies, e.g. human transferrin; tetranectins, e.g. monomeric or trimeric human C-type lectin domain; microbodies, e.g. trypsin-inhibitor-II; an armadillo repeat protein; tetraspanins, B-type lection domain containing elements, EZH type domain containing elements, avimers, nanofitins and affilins.

5. The antigen binding protein of any one of the preceding claims, wherein the antigen binding protein has at least one of the following characteristics:12.a. binds to SCGF with a Kdof less than 1 nM, of less than 100 pM, of less than 10 pM, or of less than 5 pM; and / or13.b. blocks the binding of SCGF to a cell responsive to SCGF with an IC50of less than 1 nM, preferably less than 200 pM; and / or14.c. triggering cytokine changes in tumor-associated macrophages with an EC50of 0.1 – 1 μg / ml in vitro6. The antigen binding protein of any of claims 1 to 5, further comprising at least one compound selected from:16.(i) a second antigen binding protein specifically binding to a target different from SCGF, preferably to the ectodomain of a different protein than SCGF, wherein the target different from SCGF is preferably an immune checkpoint protein (such as PD1, PDL-1 or CTLA-4), CCR5, HGF receptor, or an IGF-1 receptor;17.(ii) a second antigen binding protein specifically binding to a SCGF receptor;18.(iii) a toxin,19.(iv) a label, preferably a radionuclide or a fluorophore;20.(v) a polypeptide comprising or consisting of a transmembrane domain and, optionally an endo domain,21.(vi) a drug,22.(vii) a chemokine,23.(viii) a cytokine, (ix) an enzyme,24.(x) a component modulating serum half-life, and / or25.(xi) an Fc part of an antibody.

7. The antigen binding protein of any one of the preceding claims, wherein the antigen binding protein comprises an scFv fragment being fused to an Fc part, wherein the Fc part is preferably a human IgGl or mouse IgG2 Fc part and / or a genetically engineered Fc part that abrogates binding of Fc receptors.

8. A nucleic acid molecule or a set of two nucleic acid molecules encoding the antigen binding protein of any one of the preceding claims.

9. A recombinant expression vector or a set of two recombinant expression vectors comprising a nucleic acid molecule(s) according to claim 8.

10. A host cell comprising the vector or set of two vectors of claim 9.

11. A method of producing the antigen binding protein of any one of claims 1-7, comprising the step of preparing said antigen binding protein from a host cell expressing said antigen binding protein.

12. A pharmaceutical composition comprising at least one antigen binding protein according to any one of claims 1 to 7, the nucleic acid according to claim 8, the vector according to claim 9, the host cell of claim 10, or a combination thereof and optionally further comprising a pharmaceutically acceptable excipient or diluent.

13. The pharmaceutical composition of claim 12 further comprising one or more compounds being selected from33.(ix) an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is preferably an antibody or a small molecule against PD1, PDL-1 or CTLA-4 and is most preferably Nivolumab,34.(x) an inhibitor of the chemokine receptor CCR5, wherein the CCR5 inhibitor is preferably Maraviroc, (xi) anti-tumor lymphocytes, wherein the anti-tumor lymphocytes are preferably chimeric antigen receptor T-cells (CAR T-cells), T-cell-receptor-engineered T-cells (TCR T- cells), chimeric antigen receptor NK-cells (CAR NK-cells), NK cell receptor- engineered NK cells (NCR NK-cells), TCR / CAR hybrid T-cells, NCR / CAR hybrid NK-cells or tumor-infiltrating lymphocytes (TILs), and35.(xii) a tyrosine kinase inhibitor, wherein the tyrosine kinase inhibitor is preferably an inhibitor of the HGF receptor and is most preferably Capmatinib.

14. At least one antigen binding protein according to any one of claims 1 to 7, the nucleic acid according to claim 8, the vector according to claim 9, or the host cell of claim 10, the pharmaceutical composition of claim 12 or 13, or a combination thereof and optionally in addition one of the further compounds being selected from items (i) to (iv) of claim 13 for use in treating or preventing a disease, wherein the disease is preferably a cancer, an inflammatory disease and / or a trauma in a subject, wherein the subject is preferably an adult.

15. The at least one antigen binding protein, the nucleic acid according, the vector, the host cell, the pharmaceutical composition, or the combination thereof for use according to claim 14, wherein the cancer is resistant to standard therapy, preferably, wherein the cancer is resistant to chemotherapy and / or immunotherapy and / or radiation therapy.

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