Hermanson G. Bioconjugate Techniques, Second Edition

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740 18. Discrete PEG Reagents

used with a reactive group on one end and a blocked hydroxyl group on the other end (e.g., as

the methyl ether). In addition, large, branched PEG molecules have been created to add more

bulk or exclusion volume at a modiﬁ cation site, thus increasing the protective effect of the PEG

molecule toward the biological molecule.

Discrete PEG compounds also have been developed in various reactive forms with methyl

ether blocking groups on the terminal end (Thermo Fisher, Quanta BioDesign). Unlike the

original PEG polymer reagents that display polydispersity, these PEG compounds are pure and

consist of only one chain length per reagent type. The chain lengths in discrete PEG modiﬁ -

ers include, for example, polyethylene oxide repeat units of 3, 4, 8, 12, and 24. NHS–mPEG

modiﬁ cation reagents can be used directly to couple with amine-containing molecules or pro-

teins through amide bond formation. This reaction occurs in aqueous buffers at physiological

pH or slightly alkaline pH conditions. Conversely, maleimide–mPEG

reagents are designed to

conjugate with thiol-containing molecules, and these may be used to target reduced disulﬁ de

bonds in proteins or thiols created on molecules using a thiolation reagent.

In addition, branched chain compounds have been developed consisting of a functional

group or a reactive group followed by a PEG

chain, which then leads to three branches each

having an mPEG

arm on them. Such compounds are expected to provide large exclusion vol-

umes in aqueous solution to surround, protect, and solubilize modiﬁ ed molecules.

Another type of PEG modiﬁ cation reagent that has been developed contains a functional

group on each end that can be used for conjugation purposes and which can be used to build

structures on solid supports, surfaces, or on other molecules. Some of these reagents have been

developed to contain an amine group on one end and a carboxylate on the other end. Such PEG-

based amino acids can be used as spacer arms to mask surfaces or provide highly hydrophilic

tethers for the attachment of afﬁ nity ligands. A thiol-PEG

-carboxylate, for instance, can be used

to modify metal particles or surfaces through dative binding of the thiol to the metal and then

create PEG–carboxylic acids for further conjugation.

Figures 18.24 through 18.26 illustrate these PEG-based modiﬁ cation reagents. The meth-

ods for their use follow the same general protocol guidelines as discussed in previous sections

Figure 18.23 Biotin–PEG

–benzophenone is a water-soluble photoreactive biotinylation reagent that can be

used to add a biotin group to surfaces or molecules containing no easily derivatized functional groups.

Figure 18.24 Discrete PEGylation reagents are available to provide a range of different chain lengths for adding

mPEG modiﬁ cation arms to biomolecules. They also can be used to add water-soluble mPEG groups to organic

molecules that are normally not very soluble in aqueous solution. The NHS ester end of the mPEG compounds

reacts with amine-containing molecules to form amide bonds, leaving the mPEG chain to interact with the

aqueous environment.

Figure 18.25 Amino-PEG

-carboxylate compounds contain a primary amine on one end and a carboxylate

group on the other end. They can be used to add water-soluble spacer arms to molecules or surfaces. Using an

amine-reactive group, the amino-PEG

-carboxylate compound can be coupled via an amide bond, thus leaving

the carboxylate end free for further conjugation reactions. Avoid the use of single-step EDC conjugation reac-

tions, as this will polymerize the amino-PEG

-carboxylate by reacting with both ends.

4. Discrete PEG Modiﬁ cation Reagents 741

742 18. Discrete PEG Reagents

Figure 18.26

The branched PEGylation compound NHS-dPEG

-(mPEG

)

contains three mPEG arms, which

provide an increased sphere of hydration around modiﬁ ed molecules compared to straight-chain PEGylation

compounds.

of this chapter for the corresponding reactive group or functional group. When modifying

biomolecules with mPEG-based reagents, a series of modiﬁ cation levels should be investigated

to determine the optimal performance in an intended application. For surface or particle modi-

ﬁ cation, reference should be made to Chapter 14, especially the section on covalent coupling to

particles.

The technology of bioconjugation has affected every conceivable

discipline in the life sciences. The application of a myriad of available

chemical reactions and reagent systems for creating novel complexes

with unique activities has made possible the assay of minute quantities

of substances, the in vivo targeting of molecules, and the modulation

of speciﬁ c biological processes. Modiﬁ ed or conjugated molecules also

have been used for puriﬁ cation, detection, or location of speciﬁ c sub-

stances, and in the treatment of disease.

Crosslinking and modifying agents can be applied to alter the native

state and function of peptides and proteins, sugars and polysaccha-

rides, nucleic acids and oligonucleotides, lipids, and almost any other

molecule imaginable that can be chemically derivatized. Through care-

ful modiﬁ cation or conjugation strategies, the structure and function of

proteins can be investigated, active site conformation discovered, or

receptor–ligand interactions revealed. Some of these techniques are

so well characterized and standardized that general protocols can be

used with broad application and with excellent prospects for success.

The following sections describe how to prepare modiﬁ ed or conjugated

biological macromolecules for use in speciﬁ c applications. The chosen

applications represent some of the most popular uses of these reagent

systems, but are by no means exhaustive.

PART III

Bioconjugate

Applications

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745

This chapter describes the design, preparation, and use of hapten–carrier conjugates used to

elicit an immune response toward a coupled hapten. The chemical reactions discussed for these

conjugations are useful for coupling peptides, proteins, carbohydrates, oligonucleotides, and

other small organic molecules to various carrier macromolecules. The resultant conjugates are

important in antibody production, immune response research, and in the creation of vaccines.

1. The Basis of Immunity

The essence of adaptive immunity is the ability of an organism to react to the presence of for-

eign substances and produce components (antibodies and cells) capable of speciﬁ cally inter-

acting with and protecting the host from their invasion. An “ antigen ” or “ immunogen ” is the

name given for a substance which is both able to elicit this type of immune response and also is

capable of interacting with the sensitized cells and antibodies which are manufactured against it.

The immune system has two basic components which respond to a challenge of a foreign

substance: a cellular response mediated by T lymphocytes and a humoral response mediated

by secreted proteins called antibodies produced by B-lymphocytes, also called plasma cells. The

B-lymphocytes recognize antigens through cell-surface immunoglobulins that bind to discrete

chemical and structural epitopes on the antigen molecule. Each B cell possesses surface immu-

noglobulin of a single type (i.e., is monoclonal) and has a binding capability that is directed

against a discrete epitopic target.

Antigen binding by a complementary immunoglobulin molecule on the surface of B cells

starts a process of cellular internalization of the foreign substance by pinocytosis. Once inter-

nalized by endosomes, systematic processing of the antigen takes place which breaks it down

into smaller components.

At this point, the endosome may fuse with vesicles containing newly synthesized or recycling

major histocompatibility complex (MHC) antigens. Some of the partially degraded antigenic

fragments may form a complex with the MHC and be transported back to the cell surface.

There they are “ presented ” to the circulating T helper (T

) cells which contain receptors

able to bind speciﬁ cally to particular structural and chemical characteristics of the degraded

antigen–MHC complex. If a T

cell recognizes and binds to the presented antigen on the

Preparation of Hapten–Carrier

Immunogen Conjugates

746 19. Preparation of Hapten–Carrier Immunogen Conjugates

surface of the antigen presenting cells (APC), the T

cell proliferates and begins to produce

various lymphokines. Finally, the recognition and binding of the presented antigen by the T

cells, coupled with the release of lymphokines, stimulates the associated B cells to proliferate

and produce antibodies which recognizes the intact antigen (Germain, 1986; Pier et al ., 2004).

Antigens usually are macromolecules that contain distinct antigenic sites or “epitopes”,

which can be recognized and interact with the various components of the immune system. They

can exist as individual molecules composed of synthetic organic chemicals, proteins, lipopro-

teins, glycoproteins, RNA, DNA, polysaccharides—or they may be parts of cellular structures

(bacteria or fungi) or viruses (Male et al ., 1987; Harlow and Lane, 1988).

Small molecules like short peptides, although normally able to interact with the products of

an immune response, often cannot cause a response on their own. These “haptens”, as they are

called, actually are incomplete antigens, and while not able by themselves to cause immunogenic-

ity or to elicit antibody production, they can be made immunogenic by coupling them to a suit-

able carrier molecule ( Figure 19.1 ). Carriers typically are antigens of higher-molecular weight

that are able to cause an immunological response when administered in vivo .

Antibodies typically are able to recognize peptide sequences as small as 5–6 amino acids in

length. For instance, IgE auto-antibodies were found to have clinical signiﬁ cance in multiple

sclerosis by binding speciﬁ cally to short 5- and 6-amino acid epitopes on the surface of myelin

proteins (Mikol et al ., 2006).

In an immune response, antibodies are produced and secreted by the B-lymphocytes in con-

junction with the T

cells. In the majority of hapten–carrier systems, the B cells end up producing

antibodies that are speciﬁ c for both the hapten and the carrier. In these cases, the T lymphocytes

will have speciﬁ c-binding domains on the carrier, but will not recognize the hapten alone. In a

kind of synergism, the B- and T-cells cooperate to induce a hapten-speciﬁ c antibody response.

After such an immune response has taken place, if the host is subsequently challenged with only

the hapten, usually it will respond by producing hapten-speciﬁ c antibodies from memory cells

formed after the initial immunization. For a review of immunobiology (see Janeway, 2004).

Figure 19.1 Immunogens are made by the crosslinking of a hapten molecule with a carrier using a conjugation

reagent.

Synthetic haptens mimicking some critical epitopic structures on larger macromolecules are

often conjugated to carriers to create an immune response to the larger ‘ parent ’ molecule. For

instance, short peptide segments can be synthesized from the known sequence of a viral coat

protein and coupled to a carrier to induce immunogenicity toward the native virus. This type

of synthetic approach to immunogen production has become the basis of much of the current

research into the creation of vaccines.

The complete picture of the immune system is much more complex than this brief discus-

sion can justly describe. In many instances, merely creating a B cell response by using synthetic

peptide-carrier conjugates, however well designed, will not always guarantee complete protec-

tive immunity toward an intact antigen. The immune response generated by a short peptide

epitope from, say, a larger viral particle or bacterial cell may only be sufﬁ cient to generate

memory at the B cell level. In these cases, it is generally now accepted that a cytotoxic T-cell

response is a more important indicator of protective immunity. Designing peptide immunogens

with the proper epitopic binding sites for both B-cell and T-cell recognition is one of the most

challenging research areas in immunology today.

Hapten–carrier conjugates also are being used to produce highly speciﬁ c monoclonal anti-

bodies that can recognize discrete chemical epitopes on the coupled hapten. The resulting mono-

clonals often are used to investigate the epitopic structure and interactions between native

proteins. In many cases, the haptens used to generate these monoclonals are again small pep-

tide segments representing crucial antigenic sites on the surface of larger proteins. Monoclonals

developed from known peptide sequences will interact in highly deﬁ ned ways with the protein

from which the sequence originated. These antibodies then can be used, for example, as com-

petitors to the natural interactions between a receptor and its ligand. Thus, using antibodies

generated from hapten–carrier conjugates, information can be obtained as to the precise sites

of binding between macromolecules.

The preparation of hapten–carrier conjugates using peptide sequences can be control-

led to produce immunogens that generate high-afﬁ nity antibodies when administered in vivo .

Pedersen et al. (2006) determined that antibody titers increased in response to increasing the

peptide-to-carrier ratio of conjugation. However, just the opposite effect was found for gen-

erating high afﬁ nity antibodies. The lower the peptide-to-carrier conjugation ratio, the higher

the relative afﬁ nity of the antibodies produced. In addition, it also was found that coupling

peptides to the carrier through a central amino acid residue caused higher antibody titers than

using a terminal amino acid residue for conjugation. For this reason, for the preparation of

particular immunogen conjugates, several ratios and methods of conjugation may have to be

investigated to result in the optimal level and afﬁ nity of antibodies produced.

2. Types of Immunogen Carriers

The most commonly used carriers are all highly immunogenic, large molecules that are capa-

ble of imparting immunogenicity to covalently coupled haptens. Some of the more useful ones

are proteins, but other carriers may be composed of lipid bilayers (liposomes), synthetic or

natural polymers (dextran, agarose, poly-L-lysine), or synthetically designed organic molecules

(i.e., dendrimers, see Chapter 7). The criteria for a successful carrier molecule are the potential

for immunogenicity, the presence of suitable functional groups for conjugation with a hapten,

2. Types of Immunogen Carriers 747

748 19. Preparation of Hapten–Carrier Immunogen Conjugates

reasonable solubility properties even after derivatization—although this is not an absolute require-

ment, since precipitated molecules can be highly immunogenic—and lack of toxicity in vivo .

Some synthetic carriers actually are designed to have low immunogenicity on their own to

minimize the potential for antibody production against them. When a hapten is coupled to

these molecules, the immune response is directed principally toward the modiﬁ cation, not at the

carrier. This design approach guides most of the immune response toward the desired target and

minimizes the production of carrier-speciﬁ c antibodies.

2.1. Protein Carriers

The ﬁ rst carrier molecules used for immunogen conjugation were proteins. A foreign protein

administered in vivo by any one of a number of potential routes nearly assured the elicitation

of an immune response. In addition, protein carriers could be chosen to be highly soluble and

possessed of abundant functional groups that could facilitate easy conjugation with a hapten

molecule. When proteins are used as carriers in immunogen complex, the conjugates can be

injected in any animal except the animal of origin for the carrier protein itself. In other words,

the use of bovine serum albumin (BSA) would not be suitable for administration into cows,

since self-proteins would not be expected to elicit good immune responses, even when attached

with hapten molecules.

The most common carrier proteins in use today are keyhole limpet hemocyanin (KLH;

MW 4.5  10

to 1.3  10

), BSA (MW 67,000), aminoethylated (or cationized) BSA (cBSA),

thyroglobulin (MW 660,000), ovalbumin (OVA; MW 43,000), and various toxoid proteins,

including tetanus toxoid and diphtheria toxoid. Other proteins occasionally used include

myoglobin, rabbit serum albumin, immunoglobulin molecules (particularly IgG) from bovine

or mouse sera, tuberculin puriﬁ ed protein derivative, and synthetic polypeptides such as poly-

L-lysine and poly-L-glutamic acid.

KLH

Perhaps the most popular carrier protein is KLH. The hemocyanin from keyhole limpets (the

mollusk Megathura crenulata) is the oxygen-carrying protein of these primitive sea creatures.

KLH is an extremely large, multi-subunit protein that contains chelated copper of non-heme

origin. In concentrated solutions above pH 7.0, it displays a characteristic opalescent blue

color that betrays its near insolubility and copper prosthetic groups. In acidic solutions, the

blue color changes to green. At physiological pH, the protein exists in various subunit aggre-

gate states of large molecular weight. For instance, in Tris buffer at pH 7.4 it is known to

associate in ﬁ ve different aggregate forms (Senozan et al., 1981). In highly alkaline or acidic

environments, KLH disassociates into subunits (Hersckovits, 1988). The protein exhibits

increased immunogenicity when it is disassociated into subunits, probably due to exposure of

additional epitopic sites to the immune system (Bartel and Campbell, 1959). The intact pro-

tein usually creates considerable light-scattering or iridescent effects due to its size and almost

colloidal nature in aqueous solutions. Subunits of KLH that are highly soluble in aqueous solu-

tion are available commercially (Thermo Fisher, Biosyn). KLH is a frequent choice for devel-

oping immunogen conjugates, especially for the treatment of cancer (Curigliano et al., 2006;

Sabbatini and Odunsi, 2007).

Since keyhole limpets are marine creatures existing in a high-salt environment, native KLH

maintains its best stability and solubility in buffers containing at least 0.9 M NaCl (not 0.9 per-

cent). As the concentration of NaCl is decreased below about 0.6 M, the protein begins to pre-

cipitate and denature. Conjugation reactions using multi-subunit KLH, therefore, should be done

under high-salt conditions to preserve the solubility of the hapten–carrier complex. KLH used in

the form of discrete subunits does not have this requirement of high salt to maintain solubility.

Native, multi-subunit KLH also should not be frozen. Freeze-thaw effects cause extensive

denaturation and result in considerable amounts of insoluble material. Commercial prepara-

tions of native KLH are typically freeze-dried solids that no longer fully dissolve in aqueous

buffers and do not display the protein ’s typical blue color due to loss of chelated copper. The

partial denatured state of these products often makes conjugation reactions difﬁ cult.

KLH contains an abundance of functional groups available for conjugation with hapten

molecules. On a per-mole basis (using an average multi-subunit MW of 5,000,000 D), KLH

has over 2,000 amines from lysine residues, over 700 sulfhydryls from cysteine groups, and

over 1,900 tyrosines. Activation of the protein with succinimidyl-4-( N -maleimidomethyl)cyclo

hexane-1-carboxylate (SMCC) (Section 5, this chapter) typically results in 300–600 maleimide

groups per molecule for coupling to sulfhydryl-containing haptens.

The preparation of immunogen conjugates often requires the coupling of a sparingly soluble

hapten to a carrier molecule. Pre-dissolving the hapten in an organic solvent and adding an

aliquot of this solution to an aqueous reaction mixture typically is done to maintain at least

some solubility of the hapten in the conjugation solution. Dimethyl sulfoxide (DMSO) may be

used for this purpose with KLH while maintaining very good solubility characteristics of the

protein as well as the hapten. KLH is completely soluble in 50 percent (v/v) DMSO, becomes

cloudy at a level of 60 percent, and deﬁ nitely precipitates at 67 percent. Therefore, conjugation

reactions may be done by adding a volume of aqueous KLH to an equal volume of hapten dis-

solved in DMSO. Care should be taken, however, to avoid buffer salt precipitation upon addi-

tion of organic solvent.

BSA and cBSA

BSA (MW 67,000) and cationized BSA (cBSA) are highly soluble proteins containing numer-

ous functional groups suitable for conjugation. Even after extensive modiﬁ cation with hap-

ten molecules these carriers usually retain their solubility. The exception to this statement is

when hydrophobic peptides or other sparingly soluble molecules are conjugated to the pro-

teins. Modiﬁ cation of any carrier with hydrophobic haptens may cause enough masking of

the hydrophilic surface to result in precipitation. Depending on the degree of precipitation,

such conjugates often are useful in generating an immune response. To limit the production of

insoluble complexes, however, the conjugation reaction can be scaled back to reduce the level

of carrier modiﬁ cation.

BSA possesses a total of 59 lysine -amine groups (with only 30–35 of these typically avail-

able for derivatization), 1 free cysteine sulfhydryl (with an additional 17 disulﬁ des buried within

its three-dimensional structure), 19 tyrosine phenolate residues, and 17 histidine imidazole

groups. The presence of numerous carboxylate groups gives BSA its net negative charge (pl 5.1).

cBSA is prepared by modiﬁ cation of its carboxylate groups with ethylene diamine (Chapter

1, Section 4.3) ( Figure 19.2 ). Controlled aminoethylation using the water-soluble carbodiimide

EDC results in blocking many of BSA ’s aspartic and glutamic acid side chains (and possibly the

2. Types of Immunogen Carriers 749