BioDrugs DOI 10.1007/s40259-014-0091-4

LEADING ARTICLE

Multi-Specific Antibodies for Cancer Immunotherapy Ron D. Jachimowicz • Sven Borchmann Achim Rothe

•

Ó Springer International Publishing Switzerland 2014

Abstract Targeted treatment of cancer with monoclonal antibodies has added to the beneficial outcome of patients. In an attempt to improve anti-tumor activity of monoclonal antibodies, multi-specific antibodies have entered the research arena. To date, only a few multi-specific constructs have entered phase III clinical trials, in contrast to classical monoclonal antibodies, which are the standard first-line therapy in several tumor entities. In this review, we will assess selected multi-specific antibodies in preclinical and clinical development that may be new treatment options for cancer patients in the very near future. We will further evaluate therapy modalities including the timely distribution or the combination of various therapeutic approaches and assess the potential role of multispecific antibodies in cancer treatment.

Key Points Advances in antibody engineering have led to numerous possibilities in the generation of multispecific antibodies Catumaxomab is the only approved multi-specific antibody construct to date for treatment of human cancer Enhancing efficacy of multi-specific antibodies is essential for further development of targeted treatment options in clinical use

1 Introduction R. D. Jachimowicz and S. Borchmann contributed equally to this article. R. D. Jachimowicz
S. Borchmann Department I of Internal Medicine, Innate Immunity Group, University Hospital Cologne, Joseph Stelzmann Str. 9, 50937 Cologne, Germany Present Address: R. D. Jachimowicz Cologne Excellence Cluster on Cellular Stress Response in Aging-Associated Diseases, University of Cologne, 50931 Cologne, Germany R. D. Jachimowicz (&) Department I of Internal Medicine, University Hospital Cologne, Kerpener Strasse 62, 50937 Cologne, Germany e-mail: [email protected] A. Rothe Oncological Therapy Center, Buchforststr. 14, 51103 Cologne, Germany

Although monoclonal antibody therapy specifically targeting a single tumor cell antigen is in widespread clinical use, only a few mechanisms have been described that lead to anti-tumor activity after treatment with monoclonal antibodies. Among those are antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), which both lead to killing of the tumor cell [1]. In an attempt to improve anti-tumor activity of monoclonal antibodies or to overcome the need for a host immune system response, antibodies were developed that utilize a conjugation of a payload toxic to the tumor cells to the antibody. Frequently used preclinical payloads include radioisotopes, catalytic toxins, drugs, cytokines, and enzymes [2]. Despite some impressive clinical trials, targeted therapy strategies are limited in their effect by the ability of cancer cells to escape antibody efficacy by down regulating the respective pathway, up regulating of alternative pathways

R. D. Jachimowicz et al.

and cross-talk between pathways, which ultimately leads to resistance generation [3, 4]. The emerging resistance prominently occurs in systems where one epitope/one action pathway drives the force of antibodies [5]. Multispecific antibodies might help to overcome some of these problems and increase the efficacy of targeted therapy [6]. They can either simultaneously engage more than one tumor cell antigen by offering different binding sites for the respective targets, or engage immune cells that are routinely not activated by conventional monoclonal antibodies; for example, T cells that are not directly activated by Fc domains due to a lacking Fc receptor [7]. Further mechanisms of action developed for single target antibodies include tumor growth pathway inhibition, delivery of toxic or radioactive payloads, activation of apoptotic pathways, interference with angiogenesis or targeting of immunomodulatory receptors [8]. All these targets can also be used in combination by multi-specific antibodies. Recent advances in the recombinant generation of antibody fragments and engineered designs have led to numerous possibilities in the generation of multi-specific antibodies (Table 1). Some of these possibilities, such as bi-specific T-cell-engaging (BiTeÒ) antibodies, heteroconjugated antibodies, or trifunctional antibodies have now entered clinical evaluation, whilst catumaxomab has already been approved for clinical practice (Table 1). Recent reviews have focused on categorizing multispecific antibodies and explaining their development, design, and production [9–11]. In this review we will assess selected multi-specific antibodies in pre-clinical and clinical development, discuss possible target structures as well as multi-specific antibody designs, evaluate therapy modalities (e.g., combination with conventional chemotherapy), and assess the potential role of multi-specific antibodies in cancer treatment in the future.

2 Hitting the Right Target One main aspect of multi-specific antibody design is the choice of the right target or target combination. These targets can be grouped into different strategies, which come with specific advantages and disadvantages. The identification of tumor-associated cell surface antigens has led to a broad variety of targets. These might be exclusively expressed, overexpressed, or mutated on cancer cells. Therapeutic antibodies deliver their payload through interference with the ligand-receptor function, the blockage or activation of pathways, the delivery of a conjugated drug or toxin or, in terms of bi- or multi-specific antibodies, in the activation of the immune system via the Fc function or the engagement of immune effector cells such as T cells or natural killer (NK) cells. Thus, bi-specific antibody therapy

requires not only the identification of the right tumorassociated antigen, but also of a suitable target for the delivery of the therapeutic payload. Array technologies allow high throughput screening of large tumor libraries for the identification of tumor-associated antigens suitable for targeted therapy. These include hematopoietic or cell surface differentiation antigens and growth factors such as CD19, CD20, CD30, carcino embryonic antigen (CEA), epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), and others. Combining tumor angiogenesis inhibitory targets with the right tumor-associated antigen might direct multi-specific antibodies to the respective cells and allow much more aggressive local tumor effects with reduced systemic toxicity, sometimes limiting currently available interference options with the above-mentioned aspects of cancer growth. Strong systemic interference with growth pathways, for example, as performed in a phase I clinical trial combining pertuzumab and cetuximab, led to severe systemic toxicities [12]. To improve tumor specificity, tumor stroma-associated antigens could also be selected as targets for multi-specific antibodies. Aiming at the tumor stroma is an interesting approach to overcome limitations of conventional antibody therapy, such as systemic effects or poor tumor invasion due to blockage of inside tumor distribution by tumor stroma, as reviewed by Matsumura [13].

3 Finding the Right Design A wide range of different multi-specific antibody designs are currently being explored. Factors that play a role in evaluating the strengths and weaknesses of a specific design are pharmacokinetics and tumor penetration characteristics. An important factor for tumor penetration is the affinity of the antibody [14, 15]. Also, a smaller molecular size of the multi-specific antibody comes with a shorter serum half-life [16], but also helps to increase tumor penetration [17]. When it comes to multi-specific antibodies targeting activating receptors on immune effector cells, potential toxic side effects due to an overly strong activation as well as unspecific activation without the presence of tumor cells, such as the cytokine storm syndrome observed in a clinical trial of a CD28 activating monoclonal antibody [18], have to be avoided. Creating the right combination of activation level, location, and timeframe will be paramount for the successful use of immune effector cell activating antibodies. Additionally, the feasibility of large-scale production has to be considered, in order to make a certain design a possibly useful tool in the treatment of patients. Explored formats include bi- or multi-specific scFv developed by self assembly or

Multi-Specific Antibodies for Cancer Immunotherapy Table 1 Multi-specific antibodies in clinical development for cancer treatment Format

Name

Target

Reference

Indication

Phase

Additional information

Status

BiTe

BAY2010112

PSA ? CD3

NCT01723475

Prostatic neoplasms

I

In castration-resistant prostate cancer

Recruiting

BiTe

Blinatumomab (MT103)

CD19 ? CD3

NCT01466179

Acute lymphoblastic leukemia

II

Ongoing

NCT01471782

Acute lymphoblastic leukemia

I/II

Recruiting

NCT01207388

Acute lymphoblastic leukemia

II

NCT00274742

Relapsed nonHodgkin lymphoma

I

NCT00560794

Acute lymphoblastic leukemia

II

NCT01741792

Diffuse large B-cell lymphoma

II

Recruiting

NCT02000427

Relapsed/refractory Philadelphia positive B-precursor acute lymphoblastic leukemia

II

Not recruiting yet

In patients with minimal residual disease

Recruiting Completed

In patients with minimal residual disease

Ongoing

BiTe

MT110

EpCAM ? CD3

NCT00635596

EpCAM ? solid tumors

I

Recruiting

BiTe

MEDI-565 (MT111)

CEA ? CD3

NCT01284231

Gastrointestinal adenocarcinomas

I

Recruiting

Enhanced soluble TCR specific to gp100 fused to a CD scFv

IMCgp100

gp100 ? CD3

NCT01211262

Malignant melanoma

I

Recruiting

F(ab) fragment fusion

MDX 447

EGFR ? FccRI

NCT00005813

Glioblastoma multiforme

I

After surgery, in combination with activated monocytes

Completed

F(ab) fragment fusion

anti-CEA x anti-DTPA

CEA ? DTPAindium

NCT00467506

Thyroid neoplasms

II

In combination with diDTPA-131I (radioimmunotherapy)

Completed

3 fused F(ab) fragments

TF2

CEA ? Hapten

NCT00860860

Colorectal neoplasms

I

In combination with Lu177-labeled di-HSGDOTA peptide IMP288 (radioimmunotherapy)

Completed

NCT01221675

Small-cell lung cancer, CEAexpressing nonsmall-cell lung carcinoma

I/II

In combination with 177Lu-IMP-288 peptide (radioimmunotherapy)

Recruiting

NCT01273402

Metastatic colorectal cancer

I

In combination with 111In/90Y-labeled hapten-peptide (IMP288) (radioimmunotherapy)

Recruiting

Heteroconjugated and crosslinked rituximab and muromonab

CD20Bi

CD3 ? CD20

http://www. nature.com/bmt/ journal/vaop/ ncurrent/full/ bmt2013133a. html

Non-Hodgkin lymphoma

I

Antibody used to arm activated autologous T cells infused after autologous stem cell transplantation

Completed

Heteroconjugated and crosslinked cetuximab and muromonab

EGFRBi

CD3 ? EGFR

NCT01081808

EGFR ? solid tumors

I

Used to arm activated autologous T cells

Recruiting

R. D. Jachimowicz et al. Table 1 continued Format

Name

Target

Reference

Indication

Phase

Additional information

Status

scFvs fused to a diphtheria toxin

DT2219ARL

CD19 ? CD22

NCT00889408

Leukemia, lymphoma

I

Immunotoxintherapy

Recruiting

2 scFvs linked to a mutated human serum albumin

MM-111

ErbB2 ? ErbB3

NCT01097460

Advanced breast cancer

I

In combination with herceptin

Recruiting

NCT01304784

Malignancies with HER-2 gene amplification

I

In various combinations with lapatinib, trastuzumab and chemotherapy agents

Recruiting

NCT00911898

Advanced breast cancer

I

NCT00189345

Ovarian cancer, peritoneal neoplasms and fallopian tube neoplasms

II

Intraperitoneal application

Completed

NCT00464893

Gastric adenocarcinoma

II

After neoadjuvant chemotherapy and attempted curative resection

Completed

NCT00352833

Gastric adenocarcinoma

II

After attempted curative resection

Completed

NCT00836654

EpCAM ? tumors with malignant ascites

II/III

NCT00563836

Epithelial ovarian cancer

II

Perioperative administration

NCT00326885

Malignant ascites

II

Peritoneal administration

Completed

NCT00822809

Malignant ascites

III

In combination with prednisolone

Completed

NCT01065246

Malignant ascites

II

NCT01320020

Epithelial cancers

I

Intravenous application for systemic response

Terminated

NCT01784900

Gastric peritoneal carcinomatosis

I

In combination with surgical resection of the lesions

Recruiting

NCT01815528

Recurrent epithelial ovarian cancer

II

In combination with chemotherapy

Recruiting

NCT00377429

Epithelial ovarian cancer

II

After complete response to chemotherapy

Completed

NCT01504256

Gastric adenocarcinoma with peritoneal carcinomatosis

II

In combination with chemotherapy

Recruiting

NCT00452140

Advanced breast cancer

II

After progression on endocrine treatment

Terminated

NCT00522457

Advanced breast cancer

II

After progression on trastuzumab

Terminated

NCT01138579

Leukemia

I/II

In combination with donor lymphocyte infusion after allogenic stem cell transplantation

Recruiting

Trifunctional antibody

Trifunctional antibody

Trifunctional antibody

Catumaxomab

Ertumaxomab

FBTA05

EpCAM ? CD3

HER-2/ Neu ? CD3

CD3 ? CD20

Completed

Completed

Completed

Completed

CEA carcino embryonic antigen, DTPA diethylenetriamine pentaacetic acid, EGFR epidermal growth factor receptor, EpCAM epithelial cell adhesion molecule, PSA prostate-specific antigen, TCR T-cell receptor

Multi-Specific Antibodies for Cancer Immunotherapy Fig. 1 Structural comparison of engineered multi-specific antibodies. Grey/black, yellow and red bars resemble different antibody specificities, blue bars are constant regions. A variety of antibody formats are depicted, including bi-specific scFv conjugated by a linker peptide (BiTeÒ) (a) or HSA (human serum albumin) (b), scFv triplebody (c), scFv-based immunotoxin (d) Fab2 without or with coupled radionuclide (e, f) as well as Fab3 with coupled radionuclide (g), conjugated IgG (h), trifunctional antibody (TriomabÒ) (i), bi-specific, tetravalent IgG-like antibody (j), scFv-IgG conjugate (k), monoclonal T-cell receptor fused to an scFv (ImmTAC) (l), hexavalent antibodies (m), tetravalent, bi-specific scFvbased antibody (TandAbÒ) (n)

conjugated by a linker, bi- or multi-specific Fab fragments, potentially coupled to a toxin or radionuclide, trifunctional antibodies incorporating two targets as well as using the Fc function, bi-specific, tetravalent IgG-like antibodies, scFvIgG conjugates, recombinant immune receptors, and others.

4 Multi-Specific Antibodies in Clinical Development There is growing interest in multi-specific antibodies as a potent upgraded antibody technology to overcome the

limitations of conventional recombinant antibodies. Today, numerous multi-specific antibodies are in clinical trials and may provide a new generation of antibody therapeutics. In Table 1 we have summarized a selection of multi-specific antibodies currently in various stages of clinical development. A schematic representation of different multi-specific antibody formats is shown in Fig. 1. We have focused our attention on a selection of multi-specific antibodies in clinical development, with an emphasis not on comprehensiveness but on exemplarily demonstrating different approaches and designs currently used in clinical development of multi-specific antibodies.

R. D. Jachimowicz et al.

4.1 BiTeÒ Antibodies MT110 is a BiTeÒ antibody simultaneously targeting the epithelial cell adhesion molecule (EpCAM or CD326), which is frequently overexpressed on malignant cells of epithelial origin and on cancer stem cells giving rise to epithelial tumors, and the T-cell receptor (TCR) CD3 complex on T cells. BiTesÒ are created by linking an scFv directed against CD3 to an scFv directed against a specific tumor cell target by a linker sequence (Table 1; Fig. 1a). BiTesÒ targeting different tumor antigens have consistently shown the ability to redirect T cells to lyse their respective target at very low effector : target ratios and at the same time do not trigger T-cell signaling by CD3 in the absence of their target [19]. In pre-clinical studies, MT110 showed in vitro activity against pancreatic cancer cell lines. In vivo treatment with MT110 led to tumor control or regression of different pancreatic cancer cell lines [20]. In NOD/SCID (nonobese diabetic/severe combined immunodeficiency) mice, MT110 was able to prevent tumor establishment of HT-29 human colorectal carcinoma cells. Furthermore, high doses of MT110 led to regression of HT-29-derived established subcutaneous tumors [21]. Currently, MT110 is in a phase I clinical trial enrolling patients with EpCAMexpressing solid malignancies. Those include adenocarcinoma of the lung, small-cell lung cancer, gastric cancer or adenocarcinoma of the gastro-esophageal junction, colorectal cancer, hormone-refractory prostate cancer, breast cancer, and ovarian cancer. Another BiTeÒ antibody, MEDI-565, is also evaluated in phase I clinical trials for gastric adenocarcinoma (Table 1). This antibody simultaneously engages CEA on tumor cells and the TCR CD3 complex on T cells. In vitro data showed that colorectal tumor cells harvested from tumor specimens of patients resistant to conventional chemotherapy and established targeted therapies were engaged and killed by the respective patient’s T cells after simultaneous treatment with MEDI-565 [22]. Blinatumomab is a BiTeÒ antibody that is already in a further stage of development. As with other BiTesÒ, it engages the TCR CD3 complex on T cells (Table 1). Blinatumomab binds to CD19, which is expressed in a range of hematological malignancies. Early clinical trials showed convincing results such as a 78 % progression-free survival rate after a median follow-up of 405 days against minimal residual disease (MRD)-positive B-lineage acute lymphoblastic leukemia [23]. In another trial, 38 patients with nonHodgkin lymphoma received low doses of blinatumomab, which led to partial and complete responses. At the highest dose level of 0.06 mg/m2, all six treated patients showed a marked response [24]. Furthermore, in vitro studies showed a potential synergistic effect of blinatumomab and rituximab [25]. A range of clinical trials are currently under way

to further investigate clinical applications of blinatumomab in a variety of hematological malignancies. Also utilizing the BiTeÒ strategy, BAY2010112 targets the prostate-specific membrane antigen (PSMA). BAY2010112 showed strong in vitro anti-tumor activity against various prostate cancer cell lines and mediated tumor regression in vivo in xenograft studies [26]. A pilot phase I study for BAY2010112 is presently recruiting patients with castration-resistant prostate cancer (Table 1). In summary, BiTeÒ antibodies showed promising results in preclinical models in a range of malignancies, and clinical trials in patients with hematological malignancies seem to indicate efficacy and safety of BiTeÒ treatment in these disease entities. Results of clinical trials initiated for solid malignancies will show whether the BiTeÒ approach is also feasible here. Overall, probably the most intriguing feature of BiTesÒ is the ability to utilize a very strong effector mechanism of the immune system, T-cell-mediated cytotoxicity, in a tumor cell-specific fashion, thus reducing the severe systemic toxicity of uncontrolled T-cell activation. A limitation of BiTeÒ antibodies might be their short serum half life, though this could be turned into an advantage when combined with continuous intravenous infusion by portable minipumps, allowing for very good control of drug dose and giving the clinician the possibility to stop the infusion and have the drug eliminated from the patient’s body within hours in case of adverse effects [19]. 4.2 Conjugated Antibodies 4.2.1 Radioimmuno Antibodies Another promising approach in clinical development of multi-specific antibodies in cancer immunotherapy is the use of targeted radioimmunotherapy. In the examples discussed below, a strategy is adopted in which a bi-specific molecule binds to a tumor antigen and provides a binding site for a molecule delivering a radioactive payload simultaneously. One example for this approach is a Fab fragment fusion antibody bi-specific for CEA and diethylenetriamine pentaacetic acid (DTPA)-indium. After binding of the antibody to CEA-expressing target cells, a 131I-di-DTPA-indium hapten is used to deliver the payload (Fig. 1f). After a successful early clinical trial showing positive results mainly for patients with medullary thyroid carcinoma (MTC) [27], further trials were launched to investigate a therapeutic effect of this approach in this tumor entity (Table 1). In 29 patients with high-risk MTC (calcitonin doubling time \2 years), median overall survival was 110 months in the treatment group compared with 61 months in a matched control cohort with comparable biomarkers of disease severity [28]. Another confirmatory

Multi-Specific Antibodies for Cancer Immunotherapy

trial in 42 patients with stage IVc heavily pretreated MTC and a calcitonin doubling time of \5 years showed a median overall survival of 43.9 months after treatment [29]. A similar radioimmunotherapy approach is used in clinical trials of TF2. This bi-specific molecule is created in an innovative way using the natural binding between the regulatory subunits of cyclic adenosine monophosphate (cAMP)-dependent protein kinase and the anchoring domains of A kinase anchor proteins. Essentially, each of the binding partners is fused to a biological entity and these new structures, when combined, form a stable complex that inherits the characteristics of both biological entities [30, 31]. This technology is named the ‘dock and lock’ method and potentially allows the creation of numerous multispecific constructs. TF2 consists of three Fab fragments, two binding to CEA and one binding to the hapten histamine-succinyl-glycine (HSG) (Fig. 1g). In a recently published clinical trial in patients with metastatic colorectal cancer, TF2 was administered, followed by 111In-IMP288 or 177Lu-IMP288, which are both bound by the HSG binding domain of TF2. 111In-IMP288 showed more than 20-fold accumulation of the radioactive substance in the tumor tissue and could be used for imaging purposes. 177 Lu-IMP288 was used to deliver a toxic radioactive payload to tumor cells. This therapy seemed safe and feasible in the patient cohort consisting of 20 patients included in the trial, with grade 3–4 transient thrombocytopenia in 10 % of the patients being the most severe adverse reaction [32]. Further clinical trials using this approach are under way, recruiting patients with colorectal neoplasms, small-cell lung cancer, and CEA-expressing non-small-cell lung carcinoma (NSCLC) (Table 1). 4.2.2 Immunotoxin Antibodies An example for the use of bi-specific antibodies in the field of immunotoxin therapy is DT2219ARL. This compound consists of two scFvs recognizing CD19 and CD22 and a catalytic diphtheria toxin (DT390). By delivering the toxin to both CD19 and CD22 expressing targets it is supposed to enhance tumor cell toxicity compared with single-target immunotoxins (Fig. 1d). The compound showed remarkable anti-tumor activity in pre-clinical in vitro and in vivo models, especially when administered repetitively to mice [33, 34]. A phase I clinical trial is currently recruiting patients with relapsed or refractory CD19-positive and CD22-positive B-lineage leukemia or lymphoma. Immunoradio or immunotoxin therapeutic monoclonal antibody strategies are approved for the treatment of cutaneous T-cell lymphoma (denileukin diftitox) [35] and B-cell nonHodgkin lymphoma (ibritumomab tiuxetan) [36] (summarized in Table 1). Targeting tumor cells more specifically

with multi-specific antibodies and thus limiting systemic toxicities of immunoradio or immunotoxin therapy might further enhance the therapeutic benefit of these strategies. 4.2.3 Heteroconjugated Antibodies Yet another design to generate multi-specific antibodies is used in CD20Bi. This molecule is created by chemically heteroconjugating anti-CD20 antibody rituximab to antiCD3 antibody muromonab (Fig. 1h) [37]. Cd20Bi-armed activated autologous T cells were administered in a pilot clinical trial including three patients after autologous stem cell transplantation for non-Hodgkin lymphoma together with low dose IL-2. This approach was deemed safe and did not interfere with autologous stem cell engraftment. Furthermore, endogenous peripheral blood monocytes (PBMC) from treated patients showed a marked increase in anti-lymphoma activity in vitro (Table 1) [38]. The similarly designed multi-specific antibody EGFRBi is created by chemical heteroconjugation of anti-EGFR antibody cetuximab and anti-CD3 antibody muromonab. In pre-clinical studies, EGFRBi-armed activated T cells increased in vitro lysis of various EGFR-expressing tumor cell lines and slowed tumor progression in vivo in mouse xenograft models of pancreatic and colorectal tumors [39]. A phase I trial for patients with EGFR-positive solid tumors is recruiting participants at the moment, with first results expected this year (Table 1). The main advantage of heteroconjugated antibodies is the usage of existing antibody structures, which are already clinically developed and have an at least somewhat known safety profile, immunogenicity, and efficacy, although these features can change after heteroconjugation. Consequently, heteroconjugation techniques can be used to derive numerous bi-specific structures from known compounds. 4.3 scFv Antibodies A different approach to design a bi-specific molecule is used in MM-111. This molecule is an engineered protein consisting of two scFv binding ErbB2 and ErbB3. These scFvs are connected to a mutated human serum albumin (HSA) by short linker sequences to extend serum half-life (Fig. 1b). By blocking both ErbB2 and ErbB3 on tumor cell surfaces and formation of a complex between MM-111 and ErbB2 and ErbB3, the bi-specific molecule inhibits both growth pathways synergistically and inhibits tumor cell growth in vitro and in vivo. Interestingly, when using MM-111 in a mouse model of breast cancer in combination with lapatinib (inhibitor of a tyrosine kinase downstream of EGFR and HER2/neu) or trastuzumab (monoclonal antibody directed against HER2/neu), the molecule’s ability to

R. D. Jachimowicz et al.

inhibit tumor growth is enhanced [40]. Consequently, MM111 has entered clinical trials in combination with trastuzumab, lapatinib and various chemotherapeutic agents and as a single-agent therapy (Table 1). ImmTAC (immune mobilizing monoclonal TCR against cancer, Immunocore, Abingdon, UK) are bi-specific biologics comprising a soluble, monoclonal TCR fused to an anti-CD3 scFv (Fig. 1l). Using a TCR instead of an antibody fragment to target tumor cells, the compound IMCgp100 consists of a soluble, affinity-enhanced TCR specific for gp100, a melanoma-specific antigen, fused to a T-cell engaging anti-CD3 scFv. The engineered TCR portion of the drug targets the gp100 peptide which is presented by HLA-A2 on the surface of melanoma cells. The anti-CD3 scFv redirects T cells to melanoma cells. The molecule is currently recruiting for a clinical phase I trial in advanced malignant melanoma patients and has shown in vitro and in vivo activity in pre-clinical research (Table 1) [41, 42]. 4.4 F(ab) Fragment Fusion Antibodies The compound MDX-447 was created by cross-linking an anti-EGFR Fab and an anti-FcR Fab, thus having similar binding sites as, for example, the conventional anti-EGFR monoclonal antibody cetuximab, but in a different molecular structure. Similar molecules were created targeting different tumor antigens [43]. In a phase I clinical trial, MDX-447 was safe and tolerated but failed to elicit a meaningful anti-tumor response in 41 patients enrolled (Table 1). However, this platform technology could possibly provide improved pharmacokinetics and functionality. 4.5 Trifunctional Antibodies Catumaxomab is the furthest developed and the one and only multi-specific antibody construct that has been approved so far. Catumaxomab, a trifunctional recombinant antibody (TriomabÒ technology, TRION Pharma, Munich, Germany), received approval in the EU for the treatment of malignant ascites in April 2009 and is the first approved bi-specific antibody worldwide. It specifically targets EpCAM and CD3 and activatory Fcc type I/III receptors via the Fc region (exemplified in Fig. 1i). A pivotal phase II/III clinical trial enrolling 258 patients with recurrent symptomatic malignant ascites led to approval of intraperitoneal injection of catumaxomab for malignant ascites in epithelial cancers (Table 1). The trial showed a significant extension of the median time to next paracentesis (77 vs 13 days) and a small but significant increase in overall survival for the 66 patients with gastric cancer (71

vs 44 days) [44]. Another clinical trial showed modest antitumor activity of intraperitoneal catumaxomab in patients with platinum-resistant epithelial ovarian cancer [45]. Further analysis of patient peritoneal fluid samples helped to clarify the mechanics of treatment with catumaxomab. Some effects were a reduction of VEGF, an increase in the activation status of T cells and notably an eradication of CD133-positive and EpCAM-positive cancer stem cells [46, 47]. In a notable case report, a patient treated for malignant ascites due to metastatic breast cancer was treated with intraperitoneal catumaxomab. In addition to extending the paracentesis-free interval, the patient showed regression of liver metastasis after treatment, hinting towards a possible systemic effect [48]. In another setting, catumaxomab was applied perioperatively to gastric cancer patients undergoing surgery after neoadjuvant chemotherapy. Catumaxomab showed potent immunogeneic effects including priming of T cells specific to other tumor antigens [49]. FBTA05 is a trifunctional antibody consisting of a mouse IgG2a and a rat IgG2b chain with their respective light chains. These two parts specifically bind CD20 and CD3. Additionally, the trifunctional antibody design also provides a hybrid Fc region for interaction with Fc receptors [50]. A clinical phase I/II trial has been launched evaluating the compound in CD20-positive relapsed B-cell lymphoma after allogeneic stem cell transplantation. The compound will be administered with a donor lymphocyte infusion (DLI). Preliminary data from compassionate use showed a decrease of leukemic cells and a shrinking of lymph nodes in three out of four patients with chronic lymphocytic leukemia (CLL) and a halt of progression in one out of four patients with high-grade refractory nonHodgkin lymphoma (Table 1) [51]. Ertumaxomab is a TriomabÒ consisting of a mouse IgG2a and a rat IgG2b chain, which targets HER2/neu and CD 3. The Fc region binds activatory Fcc type I/III receptors as well, making the molecule trifunctional. Early clinical trials in advanced breast cancer showed safety with only fully reversible adverse effects and encouraging antitumor activity. Out of 15 patients evaluable, one developed a complete response, two developed a partial response, and two had stable disease [52, 53]. Building on these promising results, a phase II clinical trial confirmed safety and showed at least some anti-tumor activity. Out of 28 patients evaluable, one developed a partial regression and eight had stable disease on day 70, though the trial was stopped early due to a strategic change in the sponsor’s clinical development program and only 28 out of the 40 planned patients were enrolled [54]. Recently, a new phase I/II clinical trial started recruiting patients with Her2/neu-positive advanced solid tumors (Table 1).

Multi-Specific Antibodies for Cancer Immunotherapy

4.6 Tandem Diabodies Tandem diabodies (TandAbsÒ, Affimed, Heidelberg, Germany) are tetravalent bi-specific antibody-based constructs incorporating variable domains with two binding sites for each antigen (Fig. 1n). One specificity facilitates the recruitment of immune effector cells while the other specificity promotes binding to a tumor-associated antigen. The absence of an Fc domain avoids IgG-mediated side effects. With a molecular mass of approximately 110 kDa, which is above the first-pass renal clearance limit, these molecules exhibit an extended half-life compared with smaller scFv-based formats [55]. TandAbÒ directed against the B-cell surface marker CD19 and the T-cell-activating CD3 receptor or the NK cell-activating CD16 receptor have shown efficacy in preclinical studies. The addition of the correspondent TandAbÒ led to a several-fold increase in patients’ T- or NK-cell activation in the presence of B-cell lymphoma cells [56]. AFM13 is a highly specific TandAbÒ directed against the FccRIII-A (CD16A) receptor and the CD30 surface receptor on Hodgkin lymphoma cells. CD16A promotes the recruitment of NK cells to lyse CD30 ? Reed-Sternberg Hodgkin lymphoma cells via anti-CD30. A phase I study (ClinicalTrials.gov identifier: NCT01221571) has recently been completed and preliminary results were presented at the ISHL-9 (9thInternational Symposium on Hodgkin Lymphoma) in 2013 (personal communication).

5 Multi-Specific Antibodies in Pre-Clinical Development As in the previous section, the selection of antibodies discussed here is supposed to exemplarily show different designs and novel approaches to multi-specific antibody therapy currently in pre-clinical development. 5.1 BiTe 2.0 In an attempt to improve the shortcomings of the BiTeÒ concept, mainly altered or reduced antigen binding and potency due to the linker sequence used for connecting the scFv, another platform for the design of bi-specific molecules was developed and named dual affinity retargeting (DART). In this design, each Fv is formed by associating the VL part on one chain with the VH part on the other chain, resulting in a VLA-VHB ? VLB-VHA configuration without a linker sequence. A DART molecule targeting CD19 and CD3 showed improved T-cell activation and cytotoxicity against CD19-positive cells compared with a BiTeÒ molecule targeting the same antigens with identical sequences in vitro. The same DART also showed dose-

dependent in vivo activity in a NOD-SCID mice model of B-cell lymphoma [57]. 5.2 Hexavalent Antibodies Another interesting approach is the creation of hexavalent antibodies by using the dock and lock method to link four Fabs directed against one target to a full IgG1 antibody (Fig. 1m). Thus, the hexavalent antibody contains six Fab regions and a single Fc region. By combining different Fabs and antibodies, multi-specific molecules can be created. Three molecules were created targeting different combinations of CD20 and CD22, which were toxic on their own to various lymphoma cell lines and CLL patient specimens in vitro. Additionally, constructs having different combinations of valencies for CD20 and CD22, specifically four valencies for CD20 and two valencies for CD22 or vice versa, showed distinctively different properties and efficacies against different tumor cell lines [58]. Therefore, creating hexabodies targeting two different tumor antigens with different targeting ratios could be a feasible way to improve multi-specific antibody therapy. Molecules targeting CD20 and CD74 were toxic to mantle cell lymphoma (MCL), B-cell lymphoma and Burkittlymphoma cell lines, and patient CLL tumor samples in vitro and showed anti-tumor activity and extension of survival in a mouse model of mantle cell lymphoma. In vitro activity of CD20 and CD74 dual targeting hexabodies was superior to a combination of both parent antibodies [59]. 5.3 ScFv Antibodies The bi-specific molecule r2820 is a recombinant fusion protein of scFv against CD28 and CD20 linked by an amino acid sequence. Interestingly, r2820 is able to activate T cells in the presence of CD20-expressing cells without primary TcR/CD3 stimulus in vitro, as a primary TcR/CD3 stimulus has been shown to be essential in T-cell activation [60]. This approach to activate T cells could be used in conjunction with other methods of T-cell activation to enhance anti-tumor effects. It has been shown that activation of tumor necrosis factor (TNF) family member receptors on tumor cells by monoclonal antibodies can induce an anti-tumor effect and appears to be well tolerated [61, 62]. A bi-specific antibody targeting TNF-related apoptosis inducing ligand receptor-2 (TRAIL-R2) and lymphotoxin-beta receptor (LTbR) was engineered by fusing an scFv against LTbR to the C- or N-terminal end of a monoclonal IgG1 antibody directed against TRAIL-R2 (Fig. 1k). The C-linked compound showed anti-tumor activity in vitro and slowed tumor growth in xenograft mouse models. The anti-tumor effect

R. D. Jachimowicz et al.

in vivo was equal or superior compared with a combination treatment with both parental antibodies [63]. This might indicate synergistic effects of simultaneous targeting of multiple TNF family member receptors. Recent advances have focused on fusing scFv constructs in creating more powerful antibodies. A range of scFv triple bodies were created consisting of a polypeptide chain with three scFvs connected in a tandem fashion (Fig. 1c). In one variant, the distal scFv was directed against CD19 and the middle scFv was directed against CD16 to trigger NK-cell and macrophage activation upon binding. This triple body showed impressive activity in assays using human mononuclear cells as effectors in vitro against leukemic cell lines and primary cells from leukemia and lymphoma patients at low concentrations [64]. In a similar fashion, triple bodies with a middle scFv directed against CD16 and distal scFvs directed against CD123 and CD33 showed potent activity against acute myeloid leukemia (AML) cell lines and primary leukemia cells isolated from peripheral blood or bone marrow of AML patients [65]. Finally, another triple body directed against CD33 and CD19 and again utilizing CD16 to activate NK cells and macrophages showed anti-tumor activity against leukemia cell lines as well [66]. This design has the possibility to create a variety of combinations and arrangements of scFv directed against targets on tumor cells and immune cells to facilitate cross-linking and anti-tumor activity. Engaging immune effector cells apart from T cells has led to the generation of further antibodies. NK cells have been shown to play an important role in tumor surveillance [67]. In an approach specifically targeting NK cells, we have generated a recombinant protein consisting of an scFv directed against a tumor-associated antigen fused by a short amino acid linker sequence to ULBP2, which is a ligand for the activating NK-cell receptor NKG2D [68, 69] (immunoligands). A compound consisting of an scFv directed against CD138 connected to ULBP2 was created in this fashion targeting CD138 on multiple myeloma cells [68]. It showed strong anti-tumor effects against multiple myeloma cell lines in vitro and was able to completely abrogate tumor growth in a xenograft mouse model when applied together with peripheral blood lymphocytes (PBL). The compound also showed in vitro activity against primary myeloma cells [68]. A similar compound simultaneously targeting NKG2D via ULBP2 and PSMA via an scFv showed in vitro cytotoxicity against tumor cells when combined with purified NK cells and slowed tumor progression in a mouse xenograft model of prostate cancer when applied with PBMCs [70]. When targeting CEA via an anti-CEA scFv connected to ULBP2 targeting NKG2D, an anti-tumor effect in vitro and slowing tumor progression in vivo could also be shown in a syngeneic mouse model of colorectal cancer [71]. Moreover, ULBP2-aCEA activated

NK, NKT and T cells in vivo in the same mouse model and tumor histology showed infiltration of CD45-positive immune cells [71]. 5.4 Dual Growth Pathway Targeting Antibodies The dual targeting of two growth pathways is an interesting concept. An example for this is a bi-specific antibody, which targets EGFR and HER3 (ErbB3). In in vitro and in vivo studies, this compound also showed potent antitumor activity against erlotinib- (inhibitor of a tyrosin kinase downstream of EGFR) and cetuximab- (monoclonal antibody directed against EGFR) resistant tumor cells, showing that dual targeting is a feasible approach in overcoming resistance to single pathway inhibition [72]. In a similar approach to target two growth receptors on tumor cells, a recombinant IgG-like bi-specific tetravalent antibody directed against EGFR and insulin-like growth factor receptor (IGFR) was developed (Fig. 1j). The bispecific antibody construct inhibited tumor growth in the BxPC3 pancreatic cancer model and, interestingly, this inhibition was superior to a combination of both parental monoclonal antibodies at a comparable dose [73]. 5.5 Drug-Like Small Molecule Antibodies Another possibility for multi-specific therapies is to connect a drug-like small molecule to an antibody fragment. 2-[3-(1,3-dicarboxypropyl)ureido]pentanedioic acid (DUPA) a drug-like small molecule ligand for PSMA was conjugated to a mutant Fab targeting CD3. This compound showed good anti-tumor activity in prophylactic and treatment xenograft mouse models [74]. This technology could lead to a range of other bi-specific molecules combining ‘conventionally’ targeting pharmaceuticals with immunological targeting and is the first technology to combine a drug-like small molecule ligand to an antibody construct.

6 Mode of Action and Therapy Modalities In addition, a wide range of multi-specific antibodies currently in clinical trials or pre-clinical development use the multi-specific binding capacity of these antibodies to target other immune effector cells, such as T cells (e.g., BiTeÒ or TriomabÒ antibodies) or NK cells (e.g., TandAbÒ or immunoligands). Directly targeting immune effector cell receptors like CD3 or NKG2D might promote an adoptive immune response against tumor cells compared with ADCC only, and create a more lasting effect of the host immune system against the tumor, possibly extending to other tumor-associated antigens not targeted primarily as

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well. Another option to increase immune system activity against the tumor is hitting immunomodulatory targets, like inhibiting programmed cell death 1 receptor activation. Immunomodulation blocking this receptor with the monoclonal antibody nivolumab has provided promising results in early clinical trials [75]. Considering therapy modalities, the exact point in time of the application in the course of therapy has to be detected to achieve the maximum clinical efficacy. Different strategies could be followed. An early treatment, possibly in a neoadjuvant setting, would provide plenty of tumor mass for potential interaction of the immune system with multi-specific antibodies and might help create a vaccination-like effect. Catumaxomab was able to induce a vaccine-like effect upon treatment of patients for malignant ascites. If these findings can be confirmed with other trifunctional antibodies, treatment with multi-specific antibodies could be used like vaccines; for example, in a neoadjuvant setting in patients who undergo primary resection of a malignancy to prevent recurrence or for patients with premalignant lesions to prevent progression. In contrast, the application of multi-specific antibodies in an adjuvant setting or very late in established treatment schedules as part of a therapy consolidation could lead to the eradication of residual tumor cells. This approach was impressively demonstrated with blinatumomab as described above. In this setting, tumor mass is low and multispecific antibodies might assist re-emerging tumor cell surveillance. On the other hand, there is a potential for combination with other agents used in cancer therapy. Imaginable options here are cytokines, immune modulators, chemotherapy, radiotherapy, small molecules or other targeted therapies.

7 Conclusion Multi-specific antibodies are new generation targeted immunotherapeutics with improved characteristics. They can be engineered by linking conventional IgG formats, smaller antibody formats or receptor-based molecules. Most likely, non-antibody-based specifically binding molecules will be incorporated in multi-specific and multi-valent targeted therapy approaches. Multi-specific antibodies bind different epitopes simultaneously and thus exhibit differences in affinity and therapeutic efficacy. Synergistic or additive effects may be observed. Various strategies to deliver the therapeutic payload are a key advantage. As demonstrated, there is an extensive new drug pipeline of multi-specific antibodies, which is likely to significantly broaden the spectrum of targeted therapeutics in oncology in the near future.

Acknowledgements and Disclosures All authors declare that they have no potential conflicts of interests that are directly relevant to the content of this article.

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