Preclinical evaluation of AFM24, a novel CD16A-specific innate immune cell engager targeting EGFR-positive tumors.

Epidermal growth factor receptor (EGFR)-targeted cancer therapy such as anti-EGFR monoclonal antibodies and tyrosine kinase inhibitors have demonstrated clinical efficacy. However, there remains a medical need addressing limitations of these therapies, which include a narrow therapeutic window mainly due to skin and organ toxicity, and primary and secondary resistance mechanisms of the EGFR-signaling cascade (e.g., RAS-mutated colorectal cancer). Using the redirected optimized cell killing (ROCK®) antibody platform, we have developed AFM24, a novel bispecific, IgG1-scFv fusion antibody targeting CD16A on innate immune cells, and EGFR on tumor cells. We herein demonstrate binding of AFM24 to CD16A on natural killer (NK) cells and macrophages with KD values in the low nanomolar range and to various EGFR-expressing tumor cells. AFM24 was highly potent and effective for antibody-dependent cell-mediated cytotoxicity via NK cells, and also mediated antibody-dependent cellular phagocytosis via macrophages in vitro. Importantly, AFM24 was effective toward a variety of EGFR-expressing tumor cells, regardless of EGFR expression level and KRAS/BRAF mutational status. In vivo, AFM24 was well tolerated up to the highest dose (75 mg/kg) when administered to cynomolgus monkeys once weekly for 28 days. Notably, skin and other toxicities were not observed. A transient elevation of interleukin-6 levels was detected at all dose levels, 2-4 hours post-dose, which returned to baseline levels after 24 hours. These results emphasize the promise of bispecific innate cell engagers as an alternative cancer therapy and demonstrate the potential for AFM24 to effectively target tumors expressing varying levels of EGFR, regardless of their mutational status.Abbreviations: ADA: antidrug antibody; ADCC: antibody-dependent cell-mediated cytotoxicity; ADCP: antibody-dependent cellular phagocytosis; AUC: area under the curve; CAR: chimeric-antigen receptor; CD: Cluster of differentiation; CRC :colorectal cancer; ECD: extracellular domain; EGF: epidermal growth factorEGFR epidermal growth factor receptor; ELISA: enzyme-linked immunosorbent assay; FACS: fluorescence-activated cell sorting; Fc: fragment, crystallizableFv variable fragment; HNSCC: head and neck squamous carcinomaIL interleukinm; Ab monoclonal antibody; MOA: mechanism of action; NK :natural killer; NSCLC: non-small cell lung cancer; PBMC: peripheral blood mononuclear cell; PBS: phosphate-buffered saline; PD: pharmacodynamic; ROCK: redirected optimized cell killing; RSV: respiratory syncytial virus; SABC: specific antibody binding capacity; SD: standard deviation; TAM: tumor-associated macrophage; TKI: tyrosine kinase inhibitor; WT: wildtype.

Redirected optimized cell killing (ROCK®): A highly versatile multispecific fit-for-purpose antibody platform for engaging innate immunity.

Redirection of immune cells to efficiently eliminate tumor cells holds great promise. Natural killer cells (NK), macrophages, or T cells are specifically engaged with target cells expressing markers after infection or neoplastic transformation, resulting in their activation and subsequent killing of those targets. Multiple strategies to redirect immunity have been developed in the past two decades, but they have technical hurdles or cause undesirable side-effects, as exemplified by the T cell-based chimeric antigen receptor approaches (CAR-T therapies) or bispecific T cell engager platforms. Our first-in-class bispecific antibody redirecting innate immune cells to tumors (AFM13, a CD30/CD16A-specific innate immune cell engager) has shown signs of clinical efficacy in CD30-positive lymphomas and the potential to be safely administered, indicating a wider therapeutic window compared to T cell engaging therapies. AFM13 is the most advanced candidate from our fit-for-purpose redirected optimized cell killing (ROCK®) antibody platform, which comprises a plethora of CD16A-binding innate immune cell engagers with unique properties. Here, we discuss aspects of this modular platform, including the advantages of innate immune cell engagement over classical monoclonal antibodies and other engager concepts. We also present details on its potential to engineer a fit-for-purpose innate immune cell engager format that can be equipped with unique CD16A domains, modules that influence pharmacokinetic properties and molecular architectures that influence the activation of immune effectors, as well as tumor targeting. The ROCK® platform is aimed at the activation of innate immunity for the effective lysis of tumor cells and holds the promise of overcoming limitations of other approaches that redirect immune cells by widening the therapeutic window.

Conditional inactivation of MLH1 in thymic and naive T-cells in mice leads to a limited incidence of lymphoblastic T-cell lymphomas.

Defects in the mismatch repair system (MMR) underlie hereditary non-polyposis colorectal cancer (HNPCC)/Lynch syndrome and also a significant number of sporadic colorectal cancers. Mice carrying a null allele for the MMR gene Mlh1 are preferentially prone to the development of lymphomas of B- and T-cell origin and to a lesser extent gastrointestinal tumors. Consistent with these findings in mice, MMR defects have also been observed in sporadic and hereditary hematological malignancies. To study the role of MLH1 for lymphomagenesis in more detail, we generated a new mouse model carrying a conditional Mlh1 allele (Mlh1(flox/flox)). Mating of these mice with EIIa-Cre recombinase transgenic mice allowed the constitutive inactivation of MLH1, and the resulting Mlh1(Δex4/Δex4) mouse line displays complete MMR deficiency and a cancer predisposition phenotype similar to Mlh1−/− mice. For T-cell specific MMR inactivation we combined the Mlh1(flox/flox) allele with the Lck-Cre transgene. In the resulting Mlh1(TΔex4/TΔex4) mice, MLH1 inactivation is limited to DP/SP thymocytes and naive peripheral T-cells. The development of T-cell lymphomas in Mlh1(TΔex4/TΔex4) mice is significantly reduced compared to Mlh1−/− mice, implying that MMR functions either at very early stages during T-cell development or even earlier in lymphoid precursor cells to suppress lymphomagenesis.

Phosphorylation regulates transcriptional activity of PAX3/FKHR and reveals novel therapeutic possibilities.

Inhibition of constitutive active signaling pathways, which are a characteristic phenomenon for many tumors, can be an effective therapeutic strategy. In contrast, oncogenic transcription factors, often activated by mutational events, are in general less amenable to small-molecule inhibition despite their obvious importance as therapeutic targets. One example of this is alveolar rhabdomyosarcoma (aRMS), in which specific translocations lead to the formation of the chimeric transcription factor PAX3/FKHR. Here, we found unexpectedly that the transcriptional activity of PAX3/FKHR can be inhibited by the kinase inhibitor PKC412. This occurs via specific phosphorylation sites in the PAX3 domain, phosphorylation of which is required for efficient DNA-binding and subsequent transcriptional activity. Consequently, we show that PKC412 exerts a potent antitumorigenic potential for aRMS treatment both in vitro and in vivo. Our study suggests that posttranscriptional modifications of oncogenic transcription factors can be explored as a promising avenue for targeted cancer therapy.

B cell-specific deficiency for Smad2 in vivo leads to defects in TGF-beta-directed IgA switching and changes in B cell fate.

Smad2 is a member of the intracellular mediators that transduce signals from TGF-beta receptors and activin receptors. Targeted inactivation of Smad2 in mice leads to early lethality before gastrulation. It was shown previously that TGF-betaRII deficiency in vivo leads to defects in B cell homeostasis, Ag responsiveness, and IgA class switch recombination of B cells. To investigate the importance of Smad2-mediated signaling in B lymphocytes, we generated a B cell-specific inactivation of Smad2 in mice (bSmad2(-/-)). bSmad2(-/-) mice had normal B cell numbers in the spleen but showed a reduced population of marginal zone B cells. In contrast, B cells in Peyer's patches and peritoneal B-1a cells of bSmad2(-/-) mice were increased in numbers. bSmad2(-/-) mice showed a reduced number of surface-IgA(+) B cells and of IgA-secreting cells in Peyer's patches, decreased levels of IgA in serum, and, after immunization with a T cell-dependent Ag, a reduced IgA response. Class switch recombination to IgA was impaired in Smad2-deficient B cells, when stimulated in vitro with LPS in the presence of TGF-beta. The growth-inhibitory effects of TGF-beta in LPS-stimulated B cells were not affected in Smad2-deficient B cells. In summary, our data indicate a crucial role of Smad2 in mediating signals for the TGF-beta-directed class switch to IgA and the induction of IgA responses in vivo. Other B cell functions like growth-inhibitory signaling, which are known to be regulated by signals via the TGF-betaR, are not affected in Smad2-deficient B cells.

Transposable elements as a source of genetic innovation: expression and evolution of a family of retrotransposon-derived neogenes in mammals.

A family of functional neogenes called Mart, related to the gag gene of Sushi-like long terminal repeat retrotransposons from fish and amphibians, is present in the genome of human (11 genes) and other primates, as well as in mouse (11 genes), rat, dog (12 genes), cat, and cow. Mart genes have lost their capacity of retrotransposition through non-functionalizing rearrangements having principally affected long terminal repeats and pol open reading frame. Most Mart genes are located on the X chromosome in different mammals. Sequence database analysis suggested that Mart genes are present in opossum (marsupial), but absent from the genome of chicken. Hence, the Mart gene family might have been formed from Sushi-like retrotransposon(s) after the split of birds and mammals (310 myr ago), but before the divergence between placental mammals and marsupials (170 myr ago). RT-PCR analysis showed that at least six Mart genes are expressed during mouse embryonic development, with in situ hybridization analysis revealing rather ubiquitous expression patterns. Mart expression was also detected in adult mice, with some genes being expressed in all tissues tested, while others showed a much more restricted expression pattern. Although additional analysis will be required to establish the function of the retrotransposon-derived Mart neogenes, these observations support the evolutionary importance of retrotransposable elements as a source of genetic novelty.