Immuno-oncology for surgeons.

Cancer has traditionally been treated with surgery, cytotoxic chemotherapy and/or radiotherapy. The focus of treatment has been the mutated neoplastic cell. Critical advances in genomic and molecular techniques herald the potential for personalized treatments. Incremental breakthroughs in immunology have translated to a step-change in care by providing a mechanistic understanding of the immune system and how it may be mobilized to target cancer cells. As a result, clinical trials of immune-modifying agents have increased at an exponential rate and are revolutionizing cancer care. It is increasingly likely that the surgical oncologist will find themself caring for patients who have had immuno-oncology therapies as part of their neoadjuvant or adjuvant treatment. This review provides an update on immuno-oncology for the surgeon, covering the mechanisms of action of the agents in use. Emerging and surgically relevant toxicities are discussed, and available data on combining and sequencing cancer surgery with immuno-oncology treatments are summarized.

El paradigma del tratamiento del cáncer está evolucionando rápidamente. Tradicionalmente, el cáncer se ha tratado con cirugía, quimioterapia citotóxica y/o radioterapia, pero el foco de atención de su tratamiento se ha polarizado en las mutaciones que dan lugar a la célula neoplásica. Los progresos fundamentales en las técnicas genómicas y moleculares han precedido a los tratamientos personalizados y fármacos dirigidos a dianas terapéuticas, pero los resultados de ensayos clínicos con dichos fármacos han sido en general decepcionantes. En los últimos años, los avances rigurosos y progresivos en inmunología se han traducido en un cambio sustancial en el tratamiento al proporcionar una comprensión mecanicista de cómo interviene el sistema inmune y cómo puede movilizarse mejor para atacar a las células cancerosas. Como resultado, las indicaciones y ensayos clínicos con fármacos modificadores del sistema inmune han aumentado a un ritmo exponencial y están revolucionando el tratamiento del cáncer. Por consiguiente, cada vez es más probable que los cirujanos oncólogos se encuentren tratando pacientes que recibirán terapias inmunooncológicas (immuno-oncology, IO) como parte de los tratamientos neoadyuvantes o adyuvantes. Esta revisión describe y proporciona una guía concisa y exhaustiva de la IO, abarcando los mecanismos de acción de los fármacos en uso. Se discuten las nuevas toxicidades con relevancia para la cirugía y se resumen los datos disponibles sobre la combinación y secuencia de la cirugía oncológica con los tratamientos IO.

Sustained TL1A expression modulates effector and regulatory T-cell responses and drives intestinal goblet cell hyperplasia.

The tumor necrosis factor (TNF) superfamily protein TNF-like 1A (TL1A) is the ligand for death receptor 3 (DR3). TL1A is induced on activated dendritic cells (DCs) and its expression has been linked to human inflammatory bowel disease. To address how TL1A might influence intestinal inflammation, we generated transgenic mice that constitutively express TL1A on DCs. TL1A transgenic mice developed striking goblet cell hyperplasia in the ileum that was associated with elevated interleukin (IL)-13 levels in the small intestine. IL-13- and IL-17-producing small intestinal lamina propria T cells were increased in TL1A transgenic mice. TL1A also enhanced regulatory T (Treg) cell turnover in vivo and directly stimulated Treg cell proliferation in vitro. The presence of TL1A attenuated the ability of Treg cells to suppress conventional T cells, an effect that required DR3 signaling in either conventional T cells or Treg cells. Our findings identify mechanisms by which chronic DR3 signaling could promote pathogenesis in inflammatory bowel disease.

Characterization of four CD18 mutants in leucocyte adhesion deficient (LAD) patients with differential capacities to support expression and function of the CD11/CD18 integrins LFA-1, Mac-1 and p150,95.

Leucocyte adhesion deficiency (LAD) is a hereditary disorder caused by mutations in the CD18 (beta2 integrin) gene. Four missense mutations have been identified in three patients. CD18(A270V) supports, at a diminished level, CD11b/CD18 (Mac-1, alphaMbeta2 integrin) and CD11c/CD18 (p150,95, alphaXbeta2 integrin) expression and function but not CD11a/CD18 (LFA-1, alphaLbeta2 integrin) expression. Conversely, CD18(A341P) supports a limited level of expression and function of CD11a/CD18, but not of the other two CD11/CD18 antigens. CD18(C590R) and CD18(R593C) show a decreasing capacity to associate with the CD11a, CD11c and CD11b subunits. Transfectants expressing the CD11a/CD18 with the C590R and R593C mutations are more adhesive than transfectants expressing wild-type LFA-1, and express the reporter epitope of the monoclonal antibody 24 constitutively. Thus, the four mutations affect CD18 differently in its capacities to support CD11/CD18 expression and adhesion. These results not only provide a biochemical account for the clinical diversity of patients with leucocyte adhesion deficiency, but also offer novel insights into the structural basis of interaction between the alpha and beta subunits, which is an integral component in our understanding of integrin-mediated adhesion and its regulation.

Analysis of the oligomeric requirement for signaling by CD40 using soluble multimeric forms of its ligand, CD154.

We describe the construction of a novel soluble dodecameric form of CD154 (CD40 ligand) that is more effective than trimeric tCD154 in triggering B cell activation. Dodecameric surfactant protein (SP)-D-CD154 was more potent than tCD154 in inducing B cell proliferation over a wide range of concentrations. At saturating concentrations, the level of proliferation triggered by SP-D-CD154 was fourfold higher than that achieved with tCD154. Moreover, stimulation with dodecameric CD154 induced higher levels of the costimulatory molecules ICAM-1 and CD86. The higher activity of dodecameric CD154 when compared to trimeric CD154 is unlikely to be due to differences in their avidity for CD40, since both forms bound to CD40 strongly. Therefore, the extent of receptor clustering directly regulates signaling by CD40.

CD134L expression on dendritic cells in the mesenteric lymph nodes drives colitis in T cell-restored SCID mice.

Transfer of CD45RB(high) CD4+ T cells to immune-deficient mice in the absence of regulatory T cells leads to a Th1-mediated colitis. In this study, we show that intestinal inflammation is characterized by a 15-fold increase in the number of CD134L+ (OX40L+)-activated DC in the mesenteric lymph nodes (MLNs) compared with BALB/c mice. This was important functionally, as administration of an anti-CD134L mAb inhibited the proliferation of T cells in the MLNs as well as their expression of the gut-homing integrin alpha(4)beta(7). Most importantly, the anti-CD134L mAb completely blocked development of colitis. Surprisingly, CD134L was found to be expressed by a proportion of dendritic cells (DC) in the MLNs of unreconstituted SCID mice, suggesting that CD134L can be induced on DC in the absence of T cell-derived signals. These results indicate that some DC in the MLNs of SCID mice express an activated phenotype and that CD134L expression by these cells is involved in the development of colitis induced by T cell transfer. Accumulation of CD134L+ DC was inhibited by cotransfer of regulatory T cells, suggesting that inhibition of the accumulation of activated DC is one mechanism by which these cells prevent immune pathology.

The effects of cysteine to alanine mutations of CD18 on the expression and adhesion of the CD11/CD18 integrins.

Of the 56 cysteines in the extracellular domain of the CD18 antigen (beta2 integrin subunit), corresponding ones are not found in 12 positions in the beta4, beta7, or beta8 integrin subunits. These 12 cysteines were mutated to alanines, either singly or in pairs, in CD18. All these mutants can support the expression of all three CD11/CD18 integrins. Transfectants expressing these variant integrins are generally more adhesive than the wild-type, suggesting that the cysteine residues, perhaps by engaging in disulphide bonds, may contribute to the maintenance of the CD11/CD18 integrins in a resting state.

Effect of integrin beta 2 subunit truncations on LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) assembly, surface expression, and function.

LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) are members of the beta2 integrins involved in leukocyte function during immune and inflammatory responses. We aimed to determine a minimized beta2 subunit that forms functional LFA-1 and Mac-1. Using a series of truncated beta2 variants, we showed that the subregion Q23-D300 of the beta2 subunit is sufficient to combine with the alphaL and alphaM subunits intracellularly. However, only the beta2 variants terminating after Q444 promote cell surface expression of LFA-1 and Mac-1. Thus, the major cysteine-rich region and the three highly conserved cysteine residues at positions 445, 447, and 449 of the beta2 subunit are not required for LFA-1 and Mac-1 surface expression. The surface-expressed LFA-1 variants are constitutively active with respect to ICAM-1 adhesion and these variants express the activation reporter epitope of the mAb 24. In contrast, surface-expressed Mac-1, both the wild type and variants, require 0. 5 mM MnCl2 for adhesion to denatured BSA. These results suggest that the role of the beta2 subunit in LFA-1- and Mac-1-mediated adhesion may be different.

Expression of the H52 epitope on the beta2 subunit is dependent on its interaction with the alpha subunits of the leukocyte integrins LFA-1, Mac-1 and p150,95 and the presence of Ca2+.

Integrin-mediated adhesion is a divalent cation-dependent process. Whether divalent cations directly participate in ligand binding or exert their effects indirectly by affecting the overall structure of the integrin heterodimers is not known. In this study we describe the epitope of the mAb H52 which has been mapped to a predicted disulfide-bonded loop (C386 and C400) in the beta2 integrin subunit. In the presence of Ca2+ and Mg2+, the H52 epitope is expressed on the monomeric beta2 subunit, the LFA-1 and Mac-1 heterodimers but not on p150,95, thus implying that this epitope is masked in p150,95. However, expression of the H52 epitope on Mac-1, but not on LFA-1, or the monomeric beta2 subunit, is dependent on the presence of Ca2+, thus suggesting that the chelation of Ca2+ causes a conformational change in Mac-1 which results in the loss of the epitope. These results suggest that expression of the H52 epitope on the beta2 subunit is dependent on its interaction with the different alpha subunits. Since the epitope itself is not required for heterodimer formation nor for ligand binding, occupancy of a Ca2+ binding site(s) must therefore affect the alphabeta subunit interactions, and thus the overall conformation of Mac-1.

The role of the cysteine-rich region of the beta2 integrin subunit in the leukocyte function-associated antigen-1 (LFA-1, alphaLbeta2, CD11a/CD18) heterodimer formation and ligand binding.

The cysteine-rich region (CRR) of the beta2 integrin subunit was replaced by that of beta1 to give the chimera beta2NV1. Beta2NV1 can combine with alphaL to form a variant leukocyte-function-associated antigen (LFA)-1 on COS cell surface, suggesting that the specificity of the beta2 interaction with alphaL does not lie in the CRR. Unlike those expressing wild-type LFA-1, COS cells expressing alphaL beta2NV1 are constitutively active in intercellular adhesion molecule (ICAM)-1 adhesion. These results suggest that activation of LFA-1 involves the release of an intramolecular constraint, which is maintained, in part, by the authentic beta2 CRR.

Affinity and kinetics of the interaction between soluble trimeric OX40 ligand, a member of the tumor necrosis factor superfamily, and its receptor OX40 on activated T cells.

OX40 ligand (OX40L) and OX40 are members of the tumor necrosis factor and tumor necrosis factor receptor superfamilies, respectively. OX40L is expressed on activated B and T cells and endothelial cell lines, whereas OX40 is expressed on activated T cells. A construct for mouse OX40L was expressed as a soluble protein with domains 3 and 4 of rat CD4 as a tag (sCD4-OX40L). It formed a homotrimer as assessed by chemical cross-linking and gel filtration chromatography. Radiolabeled sCD4-OX40L bound to activated mouse T cells with a high affinity (KD = 0.2-0.4 nM) and dissociated slowly (koff = 4 x 10(-5) s-1). The affinity and kinetics of the OX40L/OX40 interactions were studied using the BIAcoreTM biosensor, which measures macromolecular interactions in real time. The extracellular part of the OX40 antigen was expressed as a soluble monomeric protein and immobilized on the BIAcore sensor chip. sCD4-OX40L bound the OX40 with a high affinity (KD = 3.8 nM), although this was lower than that determined on the surface of activated T cells (KD = 0.2-0.4 nM), where there is likely to be less restriction in mobility of the receptor. In the reverse orientation, sOX40 bound to immobilized sCD4-OX40L with a stoichiometry of 3.1 receptors to one ligand, with low affinity (KD = 190 nM) and had a relatively fast dissociation rate constant (koff = 2 x 10(-2) s-1). Thus if the OX40 receptor is cleaved by proteolysis, it will release any bound ligand and is unlikely to block re-binding of ligand to cell surface OX40 because of the low monomeric affinity.

OX40 is differentially expressed on activated rat and mouse T cells and is the sole receptor for the OX40 ligand.

OX40, a member of the tumor necrosis factor (TNF) receptor/nerve growth factor (NGF) receptor superfamily was first identified as a marker of activated rat CD4+ cells with the MRC OX40 monoclonal antibody (mAb). A ligand for OX40 (called OX40 ligand or OX40L) has recently been identified and has sequence similarity to TNF. Mouse OX40L-immunoglobulin fusion protein (OX40L-Ig) binds to activated mouse CD4+ and CD8+ cells (Baum, P. R. et al., EMBO J. 1994. 13: 3992) suggesting that OX40 could have a differential pattern of expression on mouse and rat T cells. This, however, did not rule out the presence of an alternative receptor on CD8+ cells that also binds the OX40L. We have compared the binding of the MRC OX40 mAb with that of OX40L-Ig to activated rat lymph node cells and show that both recognize the same protein, namely OX40 which is expressed on CD4+ and CD4+ CD8 alpha+ cells, but not on CD4-CD8+ cells. We have raised a new mAb (MRC OX86) using recombinant mouse OX40 protein and show by two-color flow cytometry that mouse OX40 is expressed on CD4 and CD8 single-positive cells. In addition, the new MRC OX86 mAb, unlike the MRC OX40 mAb, did not block binding of the OX40L. We conclude that OX40 is differentially expressed on activated mouse and rat T cells and is the sole receptor for the OX40L.

RNA enzymes as tools for gene ablation.

Ribozymes have the potential to ablate the expression of any gene in a sequence-specific manner and, therefore, may be useful as therapeutic molecules or as tools for the analysis of gene function. Although a number of reports have described ribozymes that are effective in inhibiting gene expression, few studies have attempted, systematically, to analyze the features of ribozymes that affect their potency within cells. Experimental observations suggest that emerging rules governing ribozyme potency in cells can be understood in terms of the competitive interactions between RNA-binding proteins, complementary RNAs and their internal secondary structure.

Synthetic polymers conjugated to monoclonal antibodies: vehicles for tumour-targeted drug delivery.

(N-(2-Hydroxypropyl)methacrylamide (HPMA)) copolymers have seen extensive development as sophisticated lysosomotropic drug carriers. They can be used for site-specific drug delivery by incorporation of appropriate targeting groups and here we report their conjugation to antitumour monoclonal antibodies (the murine IgG, antibody B72.3 and its Fab' and Fab'2 fragments) and assessment as vehicles for tumour-specific drug delivery. Conjugates were synthesised containing an average 5 copolymer units (Mw 20kD) per antibody molecule. Kinetics of elimination and body distribution of radiolabelled conjugates in mice were substantially modified compared with native antibody and fragments, showing prolonged circulation in the bloodstream. Notably, the half-time for bloodclearance of the Fab' fragment (35min) was extended ten-fold following conjugation (6h). The conjugates provoked only a low immune response in A/J mice, following three injections in adjuvant (IgG titre-1 less than 100), and were resistant (up to 50%) to proteolytic degradation by preparations of rat liver lysosomal enzymes. The parent antibody targeted efficiently to human colorectal carcinoma (LS174T) xenografts in nude mice (up to 25%/g); the conjugates, however, showed no tumour-targeting, probably due to masking by polymer chains (which are attached by non-specific aminolysis). Conjugates designed to maintain immunoreactivity following linkage through oxidised carbohydrates are currently being synthesised. Nevertheless, the conjugates display increased rates of extravasation, compared with proteins of the same hydrodynamic size, and the decreased charge is anticipated to accelerate diffusion through tumour interstitium.